Methods and compositions relating to hpv-associated pre-cancerous and cancerous growths, including cin

ABSTRACT

The present invention concerns the use of E6 and/or E7 peptides from human papilloma virus (HPV) to evaluate a cell-mediated response in a patient infected with HPV to determine the prognosis for that patient with respect to the development or recurrence of pre-cancerous or cancerous growths, including cervical intraepithelial neoplasia (CIN).

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/306,809 filed on Jul. 20, 2001, which is hereby incorporated byreference in its entirety.

The U.S. government may own rights in the present invention pursuant togrant number CA65561 and CA77378 from the National Cancer Institute andgrant number CA 16672 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of immunology,virology, and oncology. More particularly, it concerns diagnostic andtherapeutic methods related to the development and recurrence ofpre-cancerous and cancerous growths or lesions, including cervicalintraepithelial neoplasia (CIN), caused by human papilloma virus (HPV).

2. Description of Related Art

Cervical cancer is the second most common malignancy in women worldwide,accounting for 15% of all cancers diagnosed in women (Parkin et al.,1993). In the United States, cervical cancer is one of the most commonneoplasm of the female genital tract. Laboratory and epidemiologicalresearch has focused on the etiological role of some types of humanpapilloma virus (HPV) in the pathogenesis of cervical neoplasia(Brinton, 1992; Munoz et al., 1992). Overall, HPV DNA has been detectedin more than 79% of specimens of women with definite cervical disease.The most prevalent HPV type is HPV 16, which is detected in high-gradesquamous intraepithelial lesions and cancer (Lörincz et al., 1992).Results from epidemiological studies support an association betweencervical neoplasia and HPV, which is markedly stronger with HPV type 16(Morrison et al., 1991; Koutsky et al., 1992; and Munoz et al., 1992).Neoplasia is characterized by abnormal growth of cells, which oftenresults in the invasion of normal tissues, e.g., primary tumors or thespread to distant organs, e.g., metastasis.

The E6 and E7 genes of HPV 16 are frequently co-expressed and are mostabundant viral transcripts in biopsies from HPV 16 positive cervicalcarcinoma (Wettstein, 1990; Seedorf et al., 1987). There is a strongevidence that co-expression of both E6 and E7 open reading frames isnecessary and sufficient for efficient malignant transformation of avariety of mammalian cells (Munger et al., 1989). Furthermore, continuedexpression of the E6 and E7 regions of the viral genome appears to berequired to maintain the malignant phenotype (von Knebel Doeberitz etal., 1988).

While some HPV infected patients develop cervical neoplasia, others donot. Also there is a high rate of spontaneous regression observedindicating the role of host immune responses. The induction of acytotoxic T-lymphocyte (CTL) response constitutes a significant defensemechanism against viral infections; occasionally, a virus-specific CTLresponse can render full protection without a concomitant antibodyresponse (Sastry et al., 1992; Bevan, 1989; Lukacher, 1984). Based onreports in the literature describing a relation between increasedprevalence of anti-HPV antibodies, in particular those directed againstthe E7 oncoprotein, with severity of the cervical disease (Cason et al.,1992; Hamsikova et al., 1994; Jha et al., 1993), it has been suggestedthat HPV-specific humoral response may not play a protective roleagainst HPV-associated cervical neoplasia (Nakagawa et al., 1996). Onthe other hand, it has been reported that individuals with defects inCMI have an increased prevalence of HPV-associated cervical neoplasia,indicating that T cells participate in the control of HPV-associatedneoplasia in humans (Nakagawa et al., 1996; Tsukui et al.; 1996;Feltkamp et al., 1993 and Clerici et al., 1997). Decreased IL-2production and proliferative responses to mitogens such as PHA andconcanavalin-A have been observed in patients with invasive cervicalcarcinoma (Park et al., 1992). A number of in vitro and in vivostrategies have been described to identify peptides from HPV-16 E6, E7,and L1 proteins that induce T-cell activity in mice and humans (Feltkampet al., 1993; Strang et al., 1990; Tindel et al., 1991; Shepherd et al.,1992; Stauss et al., 1992; Kast et al., 1993). Typically, induction ofvirus-specific CTLs can be effected by infection with a virus orrecombinant virus that expresses a viral gene product. The viral geneproduct is processed and presented as a peptide on the surface ofinfected cells in association with an MHC class I molecule forrecognition by the CTL (Unanue, 1989).

Additionally, research efforts have concentrated on identifying andcharacterizing HIV peptides that elicit a viral-specific CTL response.Townsend et al. illustrated the concept of using T-cell epitopes inproteins as vaccine candidates when their group demonstrated the use ofshort synthetic peptides from influenza nucleoprotein as epitopes forCTL responses (Townsend et al., 1986). The inventors and others havereported using synthetic peptides to generate virus-specific CTLs invivo (Kast et al., 1991; Aichele et al., 1990; Deres et al., 1989;Sastry et al., 1992; Sastry et al., 1994; Casement et al., 1995) againstinfluenza, lymphocytic choriomeningitis, Sendai virus and HIV.

Over 90% of cervical carcinomas express human papillomavirus (HPV) E6and E7 proteins. These unique antigens are ideal targets for thedevelopment of cytotoxic T-lymphocytes (CTL) for antitumorimmunotherapy. Synthetic peptides have been identified correspondingwith the E6 and E7 oncoproteins of HPV-16 that were effective inincluding HPV-specific CTL responses in vivo (Sarkar et al., 1995).Recently, Nakagawa et al. reported that systemic T-cell proliferativeresponses and CTL responses to HPV-16 peptides and proteins weredetectable in many virgin as well as sexually active women withoutcervical lesions but not in those with active disease (Nakagawa et al.,1997). Similarly, Tsukui et al. reported that TH lymphocyte response,particularly IL-2 production, to HPV antigens was greater amongcytologically normal women than in women with different degrees ofprogressive cervical neoplasia (Tsukui et al., 1996). Also, Clerici etal. observed that production of TH1 cytokines (IL-2 and IFN-γ) whichpotentially enhances CMI, to be defective in women with extensive HPVinfection and that progression to CIN to be associated with a shift fromTH1 to TH2 cytokine production (Clerici et al., 1997). Employing a longterm in vitro stimulation protocol for determining the TH activityKadish et al. reported that lymphoproliferative responses to specificHPV peptides were associated with HPV clearance and regression to CIN(Kadish et al., 1997). On the other hand, de Gruijil et al. reportedthat T-cell proliferative responses to HPV16 E7 peptides correlated withpersistence of HPV infection, but antigen-specific IL-2 production wasassociated with both virus clearance as well as progression of cervicallesions (de Gruijil et al., 1996).

A common clinical management strategy for CIN patients includesexcisional or ablative treatment. However, follow-up studies indicatethat a significant number of patients experience recurrence. At presentno clear understanding exists regarding the development of pre-cancerousor cancerous growths, their recurrence, or disease-free status in thepatients who have undergone ablative or excisional treatment for CIN.Better and improved strategies for effective diagnostics ofHPV-associated pre-cancerous or cancerous growths and lesions is needed.

SUMMARY OF THE INVENTION

The present invention is based on the observation that a cell-mediatedimmune (CMI) response by patients infected with human papilloma virus(HPV) against E6 and/or E7 peptides of HPV is correlated with theirprognosis. A cell-mediated immune response is indicative of a reducedrisk for the development of pre-cancerous or cancerous growths in thegenitourinary tract, particularly the cervix, than the risk for apatient who does not exhibit a cell-mediated immune response; in otherwords, a patient who exhibits a positive cell-mediate immune response toparticular E6 and/or E7 peptides has a good prognosis with respect tothe development of HPV-associated pre-cancerous or cancerous growths.Alternatively, a patient who exhibits no or a low CMI response to an E6or E7 proteinaceous compound of HPV has a greater risk of a badprognosis with respect to physiological effects as a result of HPVinfection. Thus, the present invention encompasses compositions andmethods for identifying patients at risk for HPV-relatedhyperproliferative conditions, including warts, CIN, and malignantgrowths or other pre-cancerous or cancerous growths; the invention isparticularly suited to evaluating patients for recurrence of ahyperproliferative condition. As used herein, the terms “growth” and“lesion” are used interchangeably. Also, the term “pre-cancerous orcancerous growth” refers to HPV-associated growths. In addition topre-cancerous or cancerous growths or lesions on the cervix, suchgrowths or lesions may occur throughout the urogenitary tract and theyinclude perineal, vulvar and penile growths or lesions. Patients forwhom the methods may be applied include any mammals capable of HPVinfection; in some embodiments, the patient is specifically contemplatedto be a human, either male or female.

In some embodiments the present invention encompasses methods fordetermining the possibility of the development or recurrence of apre-cancerous or cancerous growth in a patient infected with humanpapilloma virus. In some cases, the patient has been treated for thegrowth. The methods involve employing the following steps: obtaining asample from the patient; incubating the sample with at least one E6 orE7 peptide of HPV; and assaying the sample for a cell-mediated immune(CMI) response against the peptide. A cell-mediated immune responseagainst an E6 or E7 peptide, or a combination thereof indicates areduced risk of recurrence as compared to a person who does not exhibitsuch a response. A pre-cancerous growth frequently observed with thedevelopment of cervical cancer is cervical intraepithelial neoplasia orCIN. In some embodiments of the invention, the method of the inventionis used with respect to patients who have or had CIN of any stage (CIN1, CIN 2, or CIN 3 or squamous intraepithelial lesions (SIL), lowgrade-SIL (L-SIL) and high grade SIL (H-SIL)). Furthermore, in otherembodiments, the methods may be implemented with patients who have orhad more severe stages of hyperproliferative growth than CIN, such as amalignancy or cancerous growth. As used in this application, the term“recurrence” refers to the appearance of a pre-cancerous or cancerousgrowth, or a re-appearance of the first growth, or evidence thereof,after a first pre-cancerous or cancerous growth was reduced, eliminated,or treated. As used herein, the term “incubating” refers to exposing orcontacting the sample with a composition that includes a peptide.

The claimed methods have applicability to human papilloma virusinfections. The human papilloma virus may be a high grade or high risktype, such as HPV 16, 18, 31, 45, 56, or 58. In some embodiments, thehuman papilloma virus is HPV 16. In other embodiments, the humanpapilloma virus is a intermediate risk type, such as HPV 33, 35, 37, 51,52, 59, 66, or 68. In still further embodiments the HPV is a low risk orlow grade type associated with warts, such as type 6, 11, 26, 40, 42,43, 44, 53, 55, 62, or 66.

The sample will include cells that give rise to a cell-mediated immuneresponse. In some embodiments, the sample is a blood sample or serumsample, while in other embodiments, the sample is obtained by lavage,smear, or swab of the area suspected of infection or known to beinfected, such as in the vaginal, cervical, or penile area. Peripheralblood mononuclear cells (PBMC) can render a cell-mediated immuneresponse and any sample containing such cells can also be employed inmethods of the invention. In some embodiments, it is contemplated thatcells from a sample are incubated in media after they have been obtainedbut before they are assayed. It is contemplated that the sample may beincubated up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours andup to 1, 2, 3, 4, 5, 6, or 7 days, and up to 1, 2, 3, 4, or 5 or moreweeks and up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months in mediaprior to assaying. It is also contemplated that the cells may beincubated in media and/or stored under conditions of sub-zero degreescentrigrade prior to assaying. The cells themselves or the cell culturesupernatant (media, not intact cells) may be used for subsequent assaysfor a cell-mediated immune response. In some embodiments, the cells areincubated between 2 and 8 hours—in some cases for 6 hours—in media priorto performing intracellular cytokine analysis by flow cytometry. Inother embodiments, the cells are incubated in media between 2 days and20 days—in some cases 15 days—in media prior to performing chromiumrelease assays to determine cytotoxic T lymphocyte (CTL) activity.

Methods of the present invention involve determining whether a patientexhibits a cell-mediated immune response against HPV peptides. Inseveral embodiments, the peptides are E6 or E7 peptides meaning theyhave an amino acid sequence that is at least 90% identical over itslength to a contiguous amino acid sequence in an E6 or E7 polypeptide.Specifically contemplated is the use of a peptide comprising 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, or more contiguous amino acids of SEQ ID NO:19(E6 from HPV 16) or SEQ ID NO:20 (E7 from HPV 16). It is contemplatedthat in some embodiments that peptides of only one sequence aretested—for example, an E6 peptide or in another example, an E7peptide—while in other embodiments, multiple sequences may be tested. Inone embodiment, an E6 and an E7 peptide are employed in methods of theinvention. In other embodiments, at least two E6 peptides (referring toat least two different E6 sequences), at least two E7 peptides(referring to at least two different E7 sequences), or both may beimplemented. It is contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,13, 14, 15 or more E6 or E7 peptides may be employed, as well as anycombination of E6 or E7 peptides thereof. In still further embodimentsthe E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, orG10S, or a combination thereof. In specific embodiments, the followingE6 peptides are employed individually or as a cocktail that includes oneor more of the following peptides: K9L, E10I, C10R, Q15L, or V10C. Whilein other embodiments, an E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V,L9V, G10C, or D20C, or a combination thereof. In certain embodiments,the following E7 peptides are employed individually or as a cocktailQ19D, R9F, R9V, L9V, G10C. Furthermore, a cocktail that includes atleast one E6 peptide and one E7 peptide from the following iscontemplated: K9L, E10I, C10R, Q15L, V10C, Q19D, R9F, R9V, L9V, or G10C.In some embodiments, it is specifically contemplated that one or morepeptides in the cocktails described above be excluded. It is alsospecifically contemplated that compositions discussed with respect todiagnostic methods of the invention may also be applied to preventativeor therapeutic methods of the invention.

Methods of the invention concern a cell-mediated immune (CMI) responseagainst human papilloma virus. There are different ways of identifyingand evaluating a cell-mediated immune response (distinguished from aserum or antibody-mediated immune response). In one embodiment of theinvention, T cell proliferation is measured. T-cell proliferation may beassayed by measuring incorporation of tritiated thymidine. Aproliferative response of equal to or greater than 2.0 using an SI scaleto at least one E6 or E7 peptide is considered positive and isindicative a patient with a reduced risk of recurrence of apre-cancerous or cancerous growth or lesion. A proliferative response ofequal to or greater than 3.0 using an SI scale to at least one E6 or E7peptide is indicative of a cell mediated response, and thus, identifiesa patient with a improved prognosis with respect to the development ofpre-cancerous or cancerous growths. Alternatively, a patient who has anSI of less than 2.0, including an SI of zero, would be considered tohave a low or no cell-mediated immune response to the E6 or E7peptide(s) and would be considered as having an increased risk for thedevelopment or recurrence of pre-cancerous or cancerous growths.

A cell mediated response can also be measured using non-radioactivemeans such as an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye or reduction assay, which is a colorimetricassay for live cells (T cell proliferation) (daCosta et al., 1999), orthe alamar Blue assay, another colorimetric assay that measuresIL-2-responding cells (Gloeckner et al., 2001; Kwack et al., 2000).

In another embodiment of the invention, assaying for a cell-mediatedimmune response involves measuring TH1 or TH2 cytokine amounts. Even ifa patient does not exhibit a CMI response by a T-cell proliferationassay, an increased risk of recurrence may be associated with theproduction of a TH2 cytokine, such as IL-10. A reduced risk ofrecurrence is observed with a patient who exhibits production of a TH1cytokine such as IFN-γ and IL-2 in response to an E6 and/or E7 peptide.In some examples, the amount of a TH1 cytokine is measured, such asIL-2, interferon (IFN) γ, tumor necrosis factor (TNF) α, or TNF-β, IL-3,IL-12, IL-15, IL-16, IL-17, or IL-18. In a specific embodiment, theamount of IL-18 is measured. In additional examples, the amount of a TH2cytokine is measured, such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,IL-10, IL-11, IL-13 or IL-14.

Measuring a CMI response may be accomplished by an immunoassay, such asELISA or a radioimmunoassay, or by flow-cytometry. In embodiments of theinvention, a sample may be assayed more than once either as duplicatesamples or by different assays. In some embodiments, more than onesample is obtained from the patient. The multiple samples may be thesame type, for example, multiple blood sample, or they may be differenttypes, for example, a blood sample and a vaginal swab.

Patients for whom the methods of the invention have applicabilityinclude patients not yet diagnosed with HPV but suspected of having HPV,patients once infected with HPV but no longer showing signs of HPVinfection, patients known to be infected with HPV, patients with apre-cancerous or cancerous growth on or around the cervix or othergenitourinary area who may or may not know she is infected with HPV,patients whose pre-cancerous or cancerous growth(s) have been treatedsuccessfully or unsuccessfully, and patients who have had at least onerecurrence of a pre-cancerous or cancerous growth(s). A pre-cancerous orcancerous growth or lesion refers to a hyperproliferative cells whosegrowth is not controlled, and includes pre-neoplasias, such as CIN andneoplasias—benign and malignant—involving squamous epithelial cells andatypical squamous cells of uncertain significance (ASCUS). It iscontemplated that a patient have more than one growth or lesion.Treatment for any growths may involve surgery—ablative or excisional—aswell as conventional cancer therapy and treatment against HPV. Suchtreatments include chemotherapy, radiation therapy, hormonal therapy,immunotherapy, administration of foscarnet, Thiovir, thiovir analogs(BioKeys), podofilox, podophyllin, trichloracetic acid (TCA), or5-fluorouracil (5-FU), intralesional or intransal interferon, Imiquimidcream. Ablative techniques include the use of liquid nitrogen,electrocautery or electrodissection, surgical excision, or lasertechnology. A successful treatment refers to treatment that completelyremoves any signs of a growth, while a partially successful treatmentrefers to a treatment that affects the growth by reducing its size orgrowth rate, or preventing its enlargement, or reducing the number ofgrowths if there is more than one. Patients once infected with HPV mayat later times not exhibits signs of an HPV infection. However, it isbelieved such patients may still experience recurrence of apre-cancerous or cancerous growth, like patients who have signs ofcontinued HPV infection.

In some methods of the invention, the patient is evaluated to determinewhether he/she is infected with HPV. In further embodiments, aserotyping of HPV is also included or is part of the initialdetermination of infection. In still further embodiments, the patient isevaluated to determine whether she has a pre-cancerous or cancerousgrowth, and if it is cancerous, whether the growth is benign ormalignant.

Methods of the invention include embodiments in which the sample isobtained from the patient at least one month after treatment for apre-cancerous or cancerous growth. The patient may have undergonetreatment for at least one pre-cancerous or cancerous growth, such as bysome form of ablation.

The present invention also includes therapeutic methods that may beemployed with the diagnostic methods of the invention. In someembodiments of the invention, a patient is identified as having anincreased risk for the development of recurrence of pre-cancerous orcancerous growths. A course of action that was not previously consideredprior to the patient being identified as having that increased risk maybe undertaken. In some embodiments, a patient who would not otherwise betreated is administered preventative treatment against pre-cancerous orcancerous growths or examined more frequently, or both. Preventativetreatments are treatments administered in the absence of physiologicalsigns of pre-cancerous or cancerous growths; “therapeutic treatment”encompasses medical treatment of a physiological condition that thepatient exhibits. These preventative treatments include the use oftherapeutic treatments for both HPV infection and HPV-associatedpre-cancerous and cancerous growths, as described above.

In some embodiments, a preventative method to protect against or reducethe risk of the development of pre-cancerous and cancerous growthsinvolves immunotherapy with HPV E6 and E7 peptides disclosed herein. Ifa patient is identified as having a low or no cell-mediated immuneresponse against a particular E6 or E7 peptide, or against a combinationof such peptides, a peptide or peptides of E6 or E7 sequence may beadministered to the patient to elicit a CMI response. Such peptidesinclude any E6 or E7 peptide, specifically including all or part of thepeptides of Table 3. Also, peptides from an E6 or E7 polypeptide, suchas those discussed with respect to diagnostic methods of the invention,may be employed in preventative methods as well. It is contemplated thatthe patient may be administered a composition containing one or morepeptide sequences, and in some embodiments, with an adjuvant,liposome-based compound, or both. In further embodiments, the patient isadministered peptides more than one time.

In some embodiments, there is a method for preventing recurrence of apre-cancerous or cancerous growth, such as CIN, in a patient infectedwith HPV and treated for the growth by identifying a patient at risk forrecurrence of an HPV-associated growth using methods disclosed herein;and, treating the patient to prevent or treat any recurrence. Treatmentoptions may involve surgery—ablative or excisional—as well asconventional cancer therapy and treatment against HPV, as describedabove. In some embodiments, the treatment is the immunotherapy treatmentinvolving at least one E6 or E7 peptide from HPV described above.

Furthermore, the present invention also encompasses kits for determiningthe possibility of recurrence of a pre-cancerous or cancer growth in apatient once infected with HPV and treated for the growth comprising, ina suitable container means, at least one E6 or E7 peptide from HPV andan antibody that allows the detection of a cell-mediated immune responseagainst the peptide. In some embodiments, the antibody is attached to anon-reacting structure on which a sample can be applied, such as a platewith wells. In further embodiments, the non-reacting structure hasmembrane, which can be affixed or attached to the structure. In someembodiments, the kit can be used in an Enzyme-Linked Immunospot(ELISPOT) Assay to detect, and in some embodiments, quantitate, cytokinesecreting cells. In still further embodiments, the kit includes anantibody against a TH1 or TH2 cytokine disclosed herein. Otherembodiments include a detection reagent to detect the included antibody.A detection reagent is any compound that allows the detection of anothercompound, including reagents that allow detection visually, such as by acolorimetric detection reagent.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-B. Proliferative responses to different E6 and E7 syntheticpeptides by PBMC from women in four different study groups. Women ingroup 1 were normal (CIN⁽⁻⁾/HPV⁽⁻⁾, n=6), in group 2 were freshlydiagnosed to be HPV-associated CIN (CIN⁽⁺⁾/HPV⁽⁺⁾), n=31), group 3 weredisease-free post-treatment (Recur⁽⁻⁾, n=22) and group 4 were withdisease recurrence (Recur⁽⁺⁾, n=10). PBMC from women in the fourdifferent groups were tested for proliferative responses to peptidesfrom the E6 or E7 oncoproteins of HPV-16. A. The stimulation index (SI)values calculated as fold increase in ³[H]thymidine incorporation inpeptide-treated samples over medium controls were shown for each patientfor each of the peptides tested. B. A summary of positive responses toE6, E7, or both peptides by each group. Group numbers are indicated onthe x-axis, while the percentage of positive responders in indicated onthe y-axis.

FIG. 2A-D. Proliferative responses of PBMC from patients in groups 3(Recur⁽⁻⁾) and 4 (Recur⁽⁺⁾) to synthetic peptides from the E6 and E7oncoproteins of HPV-16. PBMC from women in these groups were tested forproliferative responses to a control peptide (control), and 7 and 8peptides each from the E6 and E7 oncoproteins, respectively, of HPV-16.Representative data showing SI values from two patients each from group3 (panels A and B) and group 4 (panels C and D) are shown.

FIG. 3. Production of TH1 cytokines by PBMC from women in group 3 inresponse to stimulation with selected E6 and E7 peptides. PBMC fromwomen in groups 3 (Recur⁽⁻⁾) and 4 (Recur⁽⁺⁾) were cultured in thepresence of the E6 peptide Q15L and E7 peptide Q19D for two days and thesupernatant fluid in each case was analyzed for the presence of variousTH1 (IL-2, IFN-γ, IL-12) and TH2 (IL-4, IL-10) cytokines by ELISA. Theamount of each cytokine produced, after adjusting to medium controls wasshown for each of the patient tested from groups 3 and 4.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Human Papilloma Virus (HPV) infection is a major risk factor forcervical cancer, and there is an association between strong HPV-specificcell mediated immunity and less severe stages of CIN. A common clinicalmanagement strategy for CIN patients includes excisional or ablativetreatment but, follow up studies indicate that a significant number ofpatients experience recurrence. At present no clear understanding existsregarding disease recurrence or disease free status in these patients.Prognostics and treatment or preventative therapies against CIN in bothinfected, uninfected and persons who undergo a recurrence of the diseaseare critical. Many treatment therapies have been tested and implementedbut they have yet to eliminate the disease or prevent the recurrence ofthe disease. Significant number of patients experience recurrence of CINbut there are no means available to test the possibility and probabilityof recurrence.

The present invention provide a methods for determining the possibilityof recurrence of CIN as a prognostic or biomarker in a patient infectedby HPV and treated for CIN. The method involves the assay and analysisof a cell mediated immune response against peptides of HPV oncoproteinssuch as E6 and E7. The method also helps in identifying an HPV-infectedpatient at risk for recurrence of CIN. The present invention, further,makes use of targeted delivery systems, kits and immunotherapeuticmeasures to prevent the recurrence of CIN and to diagnose a patient withhigh risk of CIN.

I. HPV

Human papillomavirus (HPV) has been identified previously as animportant cofactor in the development of stages of cervical neoplasiaand cancer. Infection with HPV is however insufficient to cause cervicalcancer. Not all women infected with HPV develop cervical cancer. Womenare often treated for dysplastic cervical disease detected at the annualPap Smear. Despite the existence of Pap smear screening, epidemiologicalinvestigations continue to implicate HPV as the single greatest riskfactor for progression to cervical neoplasia and cancer. CervicalIntraepithelial Neoplasia (CIN) is a type of cervical cancer caused byHuman Papilloma Virus (HPV). HPV is associated with development ofcervical cancer, specifically HPV types 16, 18, 31, 45, 56 and 58 Thesecomprise the High Grade Type/High Risk type of HPV. Intermediategrade/risk type include HPV 33, 35, 37, 51, 52, 59, 66, and 68. OtherLow Grade Types/Low Risk Type associated with warts are types 6, 11, 26,40, 42, 43, 44, 53, 54, 55, 62, and 66. These low grade types are notmalignant in nature. The HPV genome is presented in an episomal(nonintegrated, circular) form in CIN, whereas the genome is oftenintegrated into host DNA in invasive cervical carcinoma. The E6 and E7oncoproteins, expressed by high-risk types of HPV, appear critical tothe malignant transformation of cervical squamous epithelium, as aconsequence of their ability to bind and then inactivate two importanttumor suppressor genes, p53 and retinoblastoma gene (Rb). Theinactivation of these tumor suppressor proteins appears to be a criticalcomponent of the oncogenic potential of HPV.

A. Diagnosis and Treatment of Cervical Cancer

Human Papillomavirus has been identified previously to be associatedwith the development of cervical carcinoma, a malignant condition whichappears to be preceded by several stages of cervical intraepithelialneoplasia (CIN). Despite the existence of Pap smear screening,epidemiological investigations continue to implicate HPV as the singlegreatest risk factor for progression to CIN, many investigationscontinue to search for host and/or viral markers that will help identifywomen infected with HPV who are at risk for CIN. Equally important isthe possibility of recurrence of CIN in patients who have been treated.Follow up studies in patients who have undergone excisional or ablativetreatment indicate that a significant number of patients experiencerecurrence. Therefore, it is very important to be able to evaluate thepossibility of recurrence of CIN. A prognosis of recurrence would allowa doctor to consider preventative options or therapy options.

Since the first reports linking HPV with cervical cancer appeared in theearly 1980s (zur Hausen, 1994), it has become generally accepted thathigh-risk HPV types contribute to the initiation and progression ofpreinvasive intraepithelial lesions to carcinoma. Indeed, it has beennoted that HPV infection culminates in a distinctive cytopathology inPap smears, characterized by perinuclear clearing with associatednuclear atypia (Kurman et al, 1994). These HPV changes have beencombined with mild dysplasia into the designation LSIL in the revisedBethesda terminology (Kurman and Solomon, 1994). The usefulness of HPVtesting is complicated by the fact that there is a need to distinguishbetween low-risk (L-SIL) and high-risk (H-SIL) HPV types (only thelatter pose a significant risk of association with dysplasia→carcinomaprogression), and the actual risk of progression. In the former case, anew hybrid capture test for HPV distinguishes high-risk HPV types(Sherman et al., 1995; Poijak et al, 1999; Clavel et al., 2000). DNAimage cytometry may be employed in addition to the methods of theinvention to diagnose patients at risk for cervical lesions (Lorenzatoet al., 2001).

1. Pap Smear

Over the last fifty years, Papanicolaou Smear (“Pap Smear”) has becomethe cornerstone of efforts to reduce cervical cancer mortality. PapSmear is effective because it identifies the earliest stages of cervicalcancer. Current estimates are that 60-70 million Pap Smears are done inthe U.S. each year. Pap Smear has thus become a norm in the detection ofcervical cancer. In spite of its broad acceptance in the medicalcommunity, studies indicate that Pap Smear screenings will fail todetect from 50%-80% of low grade cancerous lesions, and even 15%-30% ofhigh grade cancerous lesions.

The first step of any cytological diagnostic method is obtainingsuitable Pap smear cells for review. In a conventional Pap smear test, acytologist examines an exfoliative cell specimen, obtained by scrapingsome cells from the lining of the cervix, smearing the cells onto aslide and staining with Papanicolaou stain. The cytologist examines thestained smears for the presence of abnormal-looking cells that indicatethe presence of a malignant condition. The term “malignant condition”refers to the presence of dysplasia including adenocarcinoma in situ(AIS), invasive carcinoma (CA), neoplastic, malignant or tumor cells orthe like.

In the method of the invention an exfoliative cell specimen is obtainedfrom a patient, who may or may not harbor a malignant condition. Thespecimen may be obtained by rotating a cervical sampling device, such asa swab, spatula, or cytobrush along a portion of cervix or vaginalmucosa to obtain a cell sample. A suitable specimen will containendocervical cells with squamous and/or glandular cells.

The exfoliative cell specimen is generally smeared on the slide toprovide a thin layer of the specimen on the surface of the slide.However, the manual observation of cellular abnormalities or theautomated analysis of cytological material can be optimized by preparing“monolayers” of cells on the specimen slides. A “monolayer” is definedas substantially two-dimensional layer of uniformly distributed cellularmaterial, predominantly made up of single cells and small clusters ofcells.

When conducting Pap Smear screenings, a gynecologist collects exfoliatedcells from the surface of the cervix and places them on slides that aresent to cytologists for further examination. Cytologists then review thecells placed on the slides and look for abnormal cells. If abnormalcells are found, the Pap Smear is considered to be positive. If noabnormal cells are found, the Pap Smear is considered to be negative.Pap Smear screening is generally recognized as a practical andeconomical procedure for the early detection of cervical cancer. In thepresent invention HPV positivity was determined by Virapap/Viratypeassay (Technologies Inc., Gaithersburg, Md.).

2. Colposcopy

While the Pap Smear process is designed for initial screening,colposcopy and related procedures are generally used to confirm PapSmear abnormalities and to grade cancerous and potential cancerouslesions. Since its introduction in 1925, colposcopy has acquired widerecognition as a follow-up clinical procedure for patients identified byPap Smear screening as having possible cervical abnormalities. It isgenerally recognized that colposcopy is highly effective in evaluatingpatients with abnormal Pap Smears and has therefore become the standardof medical care in the Western world for this circumstance. It isestimated that approximately 4 million colposcopy examinations arecurrently performed in the U.S. each year.

3. Fluorescence Spectroscopy

Another method for detecting pre-cancerous and cancerous growths orlesions involves fluorescence spectroscopy, which has the capability toquickly, non-invasively and quantitatively probe the biochemical andmorphological changes that occur as tissue becomes neoplastic. Thealtered biochemical and morphological state of the neoplastic tissue isreflected in the spectral characteristics of the measured fluorescence.U.S. Pat. Nos. 6,258,576 and 6,135,965 discuss diagnosis of cervicalsquamous intraepithelial (CIN) lesions and are specifically incorporatedby reference.

4. Treatment of Precancerous and Cancerous Growth

A treatment is intended to effect an elimination, reduction orretardation of the growth. The cancerous growths can treated by excisionor ablative procedures. In addition to the immunotherapy discussed infurther detail below, the following treatments may be employedtherapeutically or preventatively in methods of the invention.

i) Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

ii) Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic or diagnosticpeptide or polynucleotide, or a chemotherapeutic or radiotherapeuticagent are delivered to a target cell or are placed in directjuxtaposition with the target cell. To achieve cell killing or stasis,both agents are delivered to a cell in a combined amount effective tokill the cell or prevent it from dividing.

iii) Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as a chimeric polypeptide of the present invention.Delivery of a chimeric polypeptide in conjuction with a second vectorencoding one of the following gene products will have a combinedanti-hyperproliferative effect on target tissues. Alternatively, asingle vector encoding both genes may be used. A variety of proteins areencompassed within the invention, including inducers of cellproliferation such as growth factor receptors, inhibitors of cellularproliferation such as tumor suppressors, and regulators of apoptosis.

iv) Ablative Procedures

A majority of persons with any cancer will generally undergo surgery ofsome type, which includes preventative, diagnostic or staging, curativeand palliative surgery. Curative surgery is a pre-cancer or cancertreatment that may be used in conjunction with other therapies, such asthe treatment of the present invention, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy and/or alternativetherapies.

Curative surgery includes resection in which all or part ofpre-cancerous or cancerous tissue is physically removed, excised, and/ordestroyed. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, and miscopicallycontrolled surgery (Mohs' surgery). It is further contemplated that thepresent invention may be used in conjunction with removal of superficialcancers, precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

v) Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abilities of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

v) Anti-Viral Agents

A patient infected with HPV may be treated with anti-viral agents aloneor in combination with anti-cancer therapies. An “anti-viral agent”refers to a composition that prevents or inhibits viral infection;prevents or inhibits the progression of a viral infection; reduces theinfectivity of the virus; prevent, inhibits, or reduces thephysiological symptoms of viral infection; prevents or reduces theincidence of viral activation; inhibits a cell that is a viral host;induces a host cell to undergo apoptosis; clears virus from all or partof the body; induces the virus to become inactive; or any combination ofthe above.

Agents used against HPV include administration of foscarnet, Thiovir,thiovir analogs (BioKeys), podofilox, podophyllin, trichloracetic acid(TCA), or 5-fluorouracil (5-FU), intralesional or intransal interferon,or Imiquimid cream. Other agents are disclosed in U.S. Pat. Nos.6,245,568, 6,238,659, and 6,214,874.

II. Proteins and Peptides Selection, Synthesis and Use

In the present invention, peptides are employed in diagnostic andtreatment methods. These peptides correspond to HPV 16 oncoproteins.

A. Proteinaceous Compositions

In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule as can beseen in peptides with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15 SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19 and in the polypeptides of SEQ ID NO: 20 and SEQ ID NO:21. As used herein, a “proteinaceous molecule,” “proteinaceouscomposition,” “proteinaceous compound,” “proteinaceous chain” or“proteinaceous material” generally refers, but is not limited to, aprotein of greater than about 200 amino acids or the full lengthendogenous sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. All the “proteinaceous” terms described above may beused interchangeably herein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900,925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250,2500 or greater amino molecule residues, and any range derivabletherein. Peptides of the invention may comprise 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or up to 100 contiguous amino acidsfrom SEQ ID NOS: 1-21, inclusive. SEQ ID NOS: 1 to 10 are peptides fromthe E6 polypeptide of HPV, while SEQ ID NOS: 11-19 are peptides from theE7 polypeptide of HPV. SEQ ID NOS: 20 and 21 are polypeptide sequencesfor HPV oncoproteins E6 and E7 respectively. The GenBank accessionnumber for E6 in HPV 16 is AF327851 (SEQ ID NO:26), while the number forE7 in HPV 16 is U76404 (SEQ ID NO:27), which are both specificallyincorporated by reference. Based on Table 3, it is understood that thepeptides specifically contemplated as part of the invention include thefollowing E6 peptides: K9L (aa 18-26 of SEQ ID NO:26), E10I (aa 23-34 ofSEQ ID NO:26), C10R (aa 37-46 of SEQ ID NO:26), Q15L (aa 43-57 of SEQ IDNO:26), V10C (aa 49-58 of SEQ ID NO:26), P9L (aa 66-74 of SEQ ID NO:26),P10I (aa 102-111 of SEQ ID NO:26), Q20P (aa 97-116 of SEQ ID NO:26),R16R (aa 131-146 of SEQ ID NO:26), G10S (aa 141-150 of SEQ ID NO:26), ora combination thereof. Based on Table 3, it is further understood thatthe peptides specifically contemplated as part of the invention includethe following E7 peptides: T10Q (aa 7-15 of SEQ ID NO:27), M9T (aa 12-20of SEQ ID NO:27), D9L (aa 14-22 of SEQ ID NO:27), Q19D (aa 44-62 of SEQID NO:27), R9F (aa 49-57 of SEQ ID NO:27), R9V (aa 66-74 of SEQ IDNO:27), L9V (aa 82-90 of SEQ ID NO:27), G10C (aa 85-94 of SEQ ID NO:27),D20C (aa 75-94 of SEQ ID NO:27), or a combination thereof.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIleallo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance which produces no significant untoward effects when appliedto, or administered to, a given organism according to the methods andamounts described herein. Organisms include, but are not limited to,Such untoward or undesirable effects are those such as significanttoxicity or adverse immunological reactions. In preferred embodiments,biocompatible protein, polypeptide or peptide containing compositionswill generally be mammalian proteins or peptides or synthetic proteinsor peptides each essentially free from toxins, pathogens and harmfulimmunogens.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or protein, polypeptide,or peptide composition that has been subjected to fractionation toremove various other proteins, polypeptides, or peptides, and whichcomposition substantially retains its activity, as may be assessed, forexample, by the protein assays, as would be known to one of ordinaryskill in the art for the specific or desired protein, polypeptide orpeptide.

It is contemplated that virtually any protein, polypeptide or peptidecontaining component may be used in the compositions and methodsdisclosed herein. However, it is preferred that the proteinaceousmaterial is biocompatible. In certain embodiments, it is envisioned thatthe formation of a more viscous composition will be advantageous in thatwill allow the composition to be more precisely or easily applied to thetissue and to be maintained in contact with the tissue throughout theprocedure. In such cases, the use of a peptide composition, or morepreferably, a polypeptide or protein composition, is contemplated.Ranges of viscosity include, but are not limited to, about 40 to about100 poise. In certain aspects, a viscosity of about 80 to about 100poise is preferred.

B. Peptides Selection, Synthesis and Use

Peptide sequences corresponding to E6 and E7 oncoproteins of HPV 16 areselected on the basis of the amphipathic structures and informationrelated to known T-cell epitopes described in literature.

The peptides of the invention can be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. Small synthetic peptide sequences,typically less than 100 residues in length, are conventionally preparedusing stepwise solid-phase synthesis. Such solid phase synthesis makesuse of an insoluble resin support for a growing oligomer. A sequence ofsubunits, destined to comprise a desired polymer, are reacted togetherin sequence on the support. A terminal amino acid is attached to thesolid support in an initial reaction, either directly or through akeying agent. The terminal residue is reacted, in sequence, with aseries of further residues such as amino acids or blocked amino acidmoieties to yield a growing oligomer attached to the solid supportthrough the terminal residue. At each stage in the synthetic scheme,unreacted reactant materials are washed out or otherwise removed fromcontact with the solid phase. The cycle is continued with a pre-selectedsequence of residues until the desired polymer has been completelysynthesized, but remains attached to the solid support. The polymer isthen cleaved from the solid support and purified for use. The foregoinggeneral synthetic scheme was developed by R. B. Merrifield for use inthe preparation of certain peptides (Merrifield, 1986). These peptidescan be synthesized either on a modified Vega 250 automatic peptidesynthesizer (Vega Biochemicals, Tucson, Ariz.) or by the “bag method” asmentioned by Houghten (Houghten, 1985). Also see, for example, Stewartand Young, (1984); Tam et al., (1983); and Barany and Merrifield (1979),each incorporated herein by reference.

Short peptide sequences, or libraries of overlapping peptides, usuallyfrom about 6 up to about 35 to 50 amino acids, which correspond to theselected regions described herein, can be readily synthesized and thenscreened in screening assays designed to identify reactive peptides.Peptides with at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or up to about 100 contiguous amino acid residues of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQID NO: 21 are contemplated by the present invention.

The compositions of the invention may include a peptide that has beenmodified to enhance its activity or to render it biologically protected.Biologically protected peptides have certain advantages over unprotectedpeptides when administered to human subjects and, as disclosed in U.S.Pat. No. 5,028,592, incorporated herein by reference, protected peptidesoften exhibit increased pharmacological activity.

Compositions for use in the present invention may also comprise peptidesthat include all L-amino acids, all D-amino acids, or a mixture thereof.The use of D-amino acids may confer additional resistance to proteasesnaturally found within the human body and are less immunogenic and cantherefore be expected to have longer biological half lives.

III. Protein Purification

Peptides and proteins derived from HPV can be purified in many ways.Generally, “purified” will refer to a specific protein, polypeptide, orpeptide composition that has been subjected to fractionation to removevarious other proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bythe protein assays, as described herein below, or as would be known toone of ordinary skill in the art for the desired protein, polypeptide orpeptide.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide, such as peptides derived from E6 and E7 oncoprotein.The term “purified protein or peptide” as used herein, is intended torefer to a composition, isolatable from other components, wherein theprotein or peptide is purified to any degree relative to itsnaturally-obtainable state. A purified protein or peptide therefore alsorefers to a protein or peptide, free from the environment in which itmay naturally occur.

Generally, “purified” will refer to a protein or peptide composition,that has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., alter pH, ionic strength, and temperature.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand also shouldprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

IV. Nucleic Acids

A. Screening of DNA

In the present invention, screening of nucleic acids may be employed notonly for screening a sample for infection but also, for detecting thepossibility of recurrence of the disease. Screening procedures whichrely on nucleic acid hybridization make it possible to isolate any genesequence from any organism, provided the appropriate probe is available.Oligonucleotide probes, which correspond to a part of the sequenceencoding the protein in question, can be synthesized chemically. Thisrequires that short, oligopeptide stretches of amino acid sequence mustbe known. The DNA sequence encoding the protein can be deduced from thegenetic code, however, the degeneracy of the code must be taken intoaccount. It is possible to perform a mixed addition reaction when thesequence is degenerate. This includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA done by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace et al., 1981). The use of aprobe or primer of between 13 and 100 nucleotides, preferably between 17and 100 nucleotides in length, or in some aspects of the invention up to1-2 kilobases or more in length, allows the formation of a duplexmolecule that is both stable and selective. Molecules havingcomplementary sequences over contiguous stretches greater than 20 basesin length are generally preferred, to increase stability and/orselectivity of the hybrid molecules obtained. One will generally preferto design nucleic acid molecules for hybridization having one or morecomplementary sequences of 20 to 30 nucleotides, or even longer wheredesired. Such fragments may be readily prepared, for example, bydirectly synthesizing the fragment by chemical means or by introducingselected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

B. Synthesis of DNA

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptides is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form.

1. Biochips

Methods of isolating arrays of biomolecules on various supports,referred to as biochips, have been developed and have been employed inDNA synthesis, sequencing, mutation studies, gene expression analysisand gene discovery. Biochips are useful in the present invention as itenables one to identify the markers for pathological states, in thiscase, HPV infection that may be of subsequent diagnostic value.

Use of a biochip involves the hybridization of a labeled molecule orpool of molecules to the targets immobilized on the biochip. The labeledmolecules are normally cDNA copies of the mRNA content of a cell ortissue. In this instance the number of copies of each distinct type ofcDNA reflects the number of copies of the corresponding. mRNA species inthe initial isolate. In general terms, the intensity of hybridization tothe target immobilized on the biochip is proportional to theconcentration of the cDNA and thus measurement of hybridizationintensity enables the relative amount of the mRNA in the initial isolateto be deduced. A relative amount of the same mRNA in two different mRNAisolated can be determined by comparing the intensities of hybridizationto the same target spot between two samples. These measurements can beused to identify markers for particular cell types or pathologicalstates that can be of subsequent diagnostic value. Alternatively, sharpincreases in the abundance of particular mRNAs in a given disease statecan indicate a possible target for drug attack, thereby providing noveltherapeutic targets.

C. Nucleic Acid Amplification Reaction

Nucleic acid molecules can be detected using a variety of techniques,including amplification reactions. The present invention contemplatesusing these amplification reactions for detecting cell mediated immuneresponse or to identify a patient who is infected with HPV and/or have aprecancerous or cancerous growth. For example, a cell-mediated immuneresponse can be detected by RT-PCR of a TH1 or TH2 cytokine disclosedherein.

1. Polymerase Chain Reaction (PCR™)

Nucleic acid used as a template for amplification is isolated from cellscontained in the biological sample, according to standard methodologies(Sambrook, 1989). The nucleic acid may be genomic DNA or fractionated orwhole cell RNA. Where RNA is used, it may be desired to convert the RNAto a cDNA.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to a K_(ATP) channel protein or a mutant thereof arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer,” as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and each incorporated herein by reference inentirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ (RT-PCR™) amplification procedure may beperformed in order to quantify the amount of mRNA amplified or toprepare cDNA from the desired mRNA. Methods of reverse transcribing RNAinto cDNA are well known and described in Sambrook et al., 1989.Alternative methods for reverse transcription utilize thermostable,RNA-dependent DNA polymerases. These methods are described in WO90/07641, filed Dec. 21, 1990, incorporated herein by reference.Polymerase chain reaction methodologies are well known in the art.

2. Other Nucleic Acid Amplification Reactions

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids that involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., PCT Application WO88/10315, incorporated herein by reference. In NASBA, the nucleic acidscan be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al. (EPA No. 329 822, incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al. (PCT Application WO 89/06700, incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, 1990, incorporated by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

D. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. Thedetection of nucleic acids may be useful in identifying a cell mediatedimmune response, a patient who is infected with HPV and/or a patient whohas a precancerous or cancerous growth.

In one embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 1989). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

HPV infection can also be detected by catalyzed signal amplifiedcolorimetric DNA in situ hybridization (CSAC-ISH) (GenPoint system,DAKO) (Birner et al., 2001).

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

V. Cell Mediated Immunity (CMI):

Some methods of the claimed invention take advantage of T-cell responsesby using them as a prognostic indicator of recurrence or as apreventative therapy against the development of CIN. More particularly,the methods assay for CMI responses to synthetic peptides from E6 and E7oncoproteins of HPV 16. The E6 and E7 genes of HPV 16 are frequentlyco-expressed and are most abundant viral transcripts in biopsies fromHPV 16 positive cervical carcinoma (Wettstein, 1990; Seedorf et al.,1987). There is a strong evidence that co-expression of both E6 and E7open reading frames is necessary and sufficient for efficient malignanttransformation of a variety of malignant transformation of a variety ofmammalian cells (Munger et al., 1989). Furthermore, continued expressionof the E6 and E7 regions of the viral genome appears to be required tomaintain the malignant phenotype (von Knebel Doeberitz et al., 1988).

Most viral infections in immune competent mammals result in acell-mediated immune response against the virus infected cells, the neteffect being lysis of the cells. During viral infections, viral proteinsare synthesized in the cell for inclusion into new viral particles. Someof those endogenous viral proteins also are degraded and transportedinto the class I antigen presentation pathway, where the foreignantigens associate with a class I MHC molecule. This peptide-MHC complexthen is transported to the surface of the cells where the foreignpeptide is presented, in the context of self MHC, to cytotoxic T cells(CTLs).

CTLs are antigen-specific effector cells. Lymphocyte surface markerstudies can be used to assay for the presence of such T-cell surfacemarkers using various procedures that are known to one of ordinary skillin the art, including the use of immunofluorescence and flow cytometry.Upon recognition of the antigen as foreign, the CTLs lyse the targetcell either through molecular interactions that induce apoptosis, orthrough secretion of pore forming enzymes that create holes in theplasma membrane disrupting its integrity. Thus, the CTL-mediated immuneresponse plays a significant role in the clearance of virally-infectedcells.

The ability of CTL effector cells to lyse virus-infected target cells isregulated by genetic and antigenic restrictions. Target cells must carrya viral antigen that is the same or equivalent to that which originallyinduced the CTLs. The target cell and the induced CTL must also bear thesame MHC class I molecule.

A. Peripheral Blood Mononuclear Cells (PBMCs)

Proliferative responses are obtained from PBMCs. There are a few methodsby which one can isolate PBMCs.

Monocytes are separated from non-rosetting cells by adherence to glassor polyethylene tissue culture vessels with or without a collagencoating in RPMI 1640 with 20% fetal calf serum (FCS), determined to befree of endotoxin, by the limulus amebocyte lysis assay; alternatively,autologous serum, or 10% AB+ normal donor serum may be used as a serumsource. Non-adherent cells are removed by gentle washing. Using thesemethods, adherent cells are usually >90% monocytes. If histologicalanalysis suggests that there is significant contamination with T cells,B cells, or NK cells, these contaminating cells were removed bytreatment with a cocktail of monoclonal antibodies, including anti-leu5b, anti-leu 12 and anti-leu11b and baby rabbit complement (Rossen etal., 1985).

Monocytes are released after 1 hr or more adherence at 37° C. in ahumidified 5% CO₂ atmosphere, for suspension culture in Teflon coatedvessels (Crowe et al., 1987). In the case of cells plated on collagencoated surfaces, 1 mg/ml collagenase type 1 is added to the medium.Cells are released by incubation, for 15 min or more in calcium andmagnesium free Dulbecco's phosphate buffered saline containing 5% FCSand EDTA. Incubations with EDTA are done on ice. A disposable cellscraper is used to help dislodge the cells. The dislodged cells arewashed ×2 in calcium and magnesium free Dulbecco's PBS and cultured inRPMI 1640 and 10% AB+ human serum in Teflong; jars as described by Croweet al. (1987).

Second strategy for isolating peripheral blood monocytes is Percolldensity gradients to enrich the monocyte concentration in thenon-rosetting population, according to Hester and Walker (1981). Themonocyte-enriched population is treated with the monoclonal antibodycocktail, described above, and complement, to remove contaminatingresidual T cells, B cells and NK cells, as necessary. Monocytes arerecovered by this method are cultured directly in Teflon coated vessels,without the ‘activation’ which necessarily occurs when monocytes becomesurface adherent. However, it is possible that the Percoll densitygradient step, and/or the exposure antibodies and complement may also‘activate’ these cells, possibly in a different manner.

A third approach to isolating peripheral blood monocytes is to takeadvantage of the retractile properties of monocytes to sort them on thebasis of forward angle light scatter, using the high speed cell sorterfunction of the flow cytometer. This alternative has the potential toproduce highly purified cells, which have not been influenced by contactwith antibody or complement.

B. T-Cell Responses

T-cell responses can be measured by a variety of protocols that areknown to one of ordinary skill in the art. Some of these assays aredescribed in fuller detail below.

1. ³[H]Thymidine Incorporation Assay

The proliferative responses of PBMCs from different samples can bedetermined by the standard ³[H]thymidine incorporation assay asdescribed in published articles (Nehete, 1996; Nehete, 1995). Thesignificance of T-cell proliferative responses to the individual E6 andE7 peptides (in terms of stimulation index [SI]) can be calculated asthe fold increase of ³[H]thymidine incorporation by cells exposed to thepeptide over that by the control to which no peptide was added. An SIvalue of at least 2.0, including at least about 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0 or more, which are considered positive responses. Generally, an SIvalue is calculated by measuring the amount of radioactivity (cpm) inmedia from cells incubated with the peptide(s) and dividing by theamount of radioactivity in media from cells not incubated withpeptide(s) (media alone).

2. Lysis Using ⁵¹[Cr]

Cell-mediated lympholysis (CML) can be used as an indication of T-cellresponse. Target cells can be labeled with radioactive chromium-51(⁵¹[Cr]) prior to exposure to effector cells. The amount of ⁵¹[Cr]released into the media is proportional to the level of cell-mediatedlysis.

3. γ-Interferon Production

Interferon gamma (γ-interferon), also called type II or immuneinterferon, is produced by T cells and NK cells. It is critical for thedevelopment of helper T cells. Because it is the primarymacrophage-activating factor, it is a strong cytokine in cell-mediatedimmunity. γ-interferon increases the levels of MHC class I and MHC classII expression, which improves antigen presentation and other cognitivereactions. Furthermore, it amplifies the effects of TNF-α and raisesexpression levels of adhesion molecules on the surface of vascularendothelial cells, which leads to T cell adhesion and extravasation.

4. Tetramer Assay

Tetramer assays are well known to those of skill in the art. See Altman,1996.

5. Cytokine Production

Cytokines are proteins that play important roles in the regulation ofimmune responses as well as in the differentiation pathways of differentcell types. They have a critical function in T cell regulation anddevelopment, and these include γ-interferon, interleukin 1 (IL-1), IL-2,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, lymphotoxin,MIF, TGF-β, TNF-α, and other chemotactic cytokines. The TH1 cytokinescomprise IL-2, interferon (IFN) γ, tumor necrosis factor (TNF) α, orTNF-β, IL-3, IL-12, IL-15, IL-16, IL-17, or IL-18. TH2 cytokines includeIL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-14 orIL-18. Assays for cytokines are well known in the art of which some aredisclosed herein

6. Cytokine Analysis

Measurement of TH1 and TH2 cytokines can be done by ELISA,Radioimmunoassay (RIA) or Flow cytometry (FACS). The steps of varioususeful immunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle and Ben-Zeev, 1999; Gulbis andGaland, 1993; and De Jager et al., 1993, each incorporated herein byreference.

7. Immunoassays

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components protein(s),polypeptide(s) or peptide(s). In some embodiments immunoassays are usedto detect a cell mediated immune response to HPV peptides. Someimmunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot to mention a few. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle MH and Ben-Zeev O, 1999; Gulbis Band Galand P, 1993; and De Jager R et al., 1993, each incorporatedherein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing protein, polypeptide and/or peptide, andcontacting the sample with an antibody in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of immunocomplexes. For example, in the present invention, E6and/or E7 peptides may be used to challenge the cells to elicit a T-cellresponse. The antibodies may be directed to cytokines produced as anoutcome of the cell mediated response or to cytokine receptors onT-cells. Alternatively, an antibody against CD69 or CD45, or both, maybe employed.

These methods include methods for purifying an protein, polypeptideand/or peptide from organelle, cell, tissue or organism's samples. Inthese instances, the antibody removes the protein, polypeptide and/orpeptide component from a sample. The antibody will preferably be linkedto a solid support, such as in the form of a column matrix, and thesample suspected of containing the protein, polypeptide and/or peptideantigenic component will be applied to the immobilized antibody. Theunwanted components will be washed from the column, leaving the antigenimmunocomplexed to the immobilized antibody to be eluted.

In terms of cytokine response detection, the biological sample analyzedmay be any sample that is suspected of containing a cytokine, such as,for example, a tissue section or specimen, a homogenized tissue extract,a cell, an organelle, separated and/or purified forms of any of theabove antigen-containing compositions, or even any biological fluid thatcomes into contact with the cell or tissue, including blood and/orserum, although tissue samples or extracts are preferred.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antigen antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. The methodprovides a useful means of identifying the population of women that areinfected with HPV having cervical cancer or CIN.

Another means of determining whether a person infected by HPV has aprecancerous growth or cancerous growth is by hybrid capture as shown inBirner et al. 2001 and Clavel et al., 2000, both incorporated byreference.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousdiseases wherein a specific is expressed, such as a cancer specific geneproduct, etc. Here, a biological and/or clinical sample suspected ofbeing infected with HPV that could lead to CIN is used. However, theseembodiments also have applications to non-clinical samples, such as inthe titering of antigen or antibody samples, for example in theselection of hybridomas.

a. ELISA

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, the antibodies directed to the product of cellmediated immune response (that comprises the antigen), in the presentinvention, are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing the antigen, such as a clinicalsample, is added to the wells. After binding and/or washing to removenon-specifically bound immune complexes, the bound antigen may bedetected. Detection is generally achieved by the addition of anotherantibody that is linked to a detectable label. This type of ELISA is asimple “sandwich ELISA”. Detection may also be achieved by the additionof a second antibody, followed by the addition of a third antibody thathas binding affinity for the second antibody, with the third antibodybeing linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withthe antibodies produced against the product of cell mediated immuneresponse (that comprises the antigen). After binding and/or washing toremove non-specifically bound immune complexes, the bound antibodies aredetected. Where the initial antibodies are linked to a detectable label,the immune complexes may be detected directly. Again, the immunecomplexes may be detected using a second antibody that has bindingaffinity for the first antibody, with the second antibody being linkedto a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst an antigen are added to the wells, allowed to bind, and/ordetected by means of their label. The amount of an antigen in an unknownsample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

b. Immunohistochemistry

The antibodies may also be used in conjunction with both fresh-frozenand/or formalin-fixed, paraffin-embedded tissue blocks prepared forstudy by immunohistochemistry (IHC). The method of preparing tissueblocks from these particulate specimens has been successfully used inprevious IHC studies of various prognostic factors, and/or is well knownto those of skill in the art (Brown et al., 1990; Abbondanzo et al.,1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

c. Fluorescent Assorted Cell Spectroscopy:

Proteins may also be detected by flow cytometry as described inFujishima et al, 1996. In the practice of the method, the cells arefixed and then incubated with a monoclonal antibody against theexpressed protein to be detected. The bound antibodies are thencontacted with labeled anti-IgG for example for detection. A typicallabel is FITC. The fluorescent intensity may then be measured by flowcytometer such as Ortho Cytron, Ortho diagnostics, or FACScan; BectonDickinson.

FACS permits the separation of sub-populations of cells initially on thebasis of their light scatter properties as they pass through a laserbeam. The forward light scatter (FALS) is related to cell size and theright angle light scatter to cell density, cell contour andnucleo-cytoplasmic ratio. Since cells are tagged with fluorescentlabeled antibody they can then be further characterized by fluorescenceintensity and positive and negative windows set on the FACS to collectbright fluorescence and low fluorescence cells. Cells are sorted at aflow rate of about 3000 cells per second and collected in positive andnegative cells.

d. Western Blots

The compositions of the present invention may find use in immunoblot orwestern blot analysis. The peptides may be used to challenge the cellsto produce cytokines. The antibodies of the present invention may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the protein moiety areconsidered to be of particular use in this regard.

VIII. Immunotherapy

Immunotherapy, as an approach to infection or cancer treatment, is basedon the premise that tumor cells bear antigens provoking the productionof specific antibodies and/or cytotoxic T lymphocytes (CTL).

A. Types of Immunotherapies:

Immunotherapies of cancer can broadly be classified as adoptive, passiveand active, as described in the following sections.

1. Passive Immunotherapy

A number of different approaches for passive immunotherapy exist. Theymay be broadly categorized into the following: injection of antibodiesalone; injection of antibodies coupled to toxins or chemotherapeuticagents; injection of antibodies coupled to radioactive isotopes;injection of anti-idiotype antibodies; and finally, purging of tumorcells in bone marrow.

Preferably, human monoclonal antibodies, are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.However, their application is somewhat limited by their scarcity andhave so far only been administered intralesionally. Human monoclonalantibodies to ganglioside antigens have been administeredintralesionally to patients suffering from cutaneous recurrent melanoma(Irie & Morton, 1986). Regression was observed in six out of tenpatients, following, daily or weekly, intralesional injections. Inanother study, moderate success was achieved from intralesionalinjections of two human monoclonal antibodies (Irie et al., 1989).

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as in Bajorin et al. (1988).The development of human monoclonal antibodies is described in furtherdetail elsewhere in the specification.

2. Active Immunotherapy

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogeneic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranath& Morton, 1991; Morton & Ravindranath, 1996; Morton et al., 1992;Mitchell et al., 1990; Mitchell et al., 1993). In melanomaimmunotherapy, those patients who elicit high IgM response often survivebetter than those who elicit no or low IgM antibodies (Morton et al.,1992). IgM antibodies are often transient antibodies and the exceptionto the rule appears to be anti-ganglioside or anticarbohydrateantibodies.

3. Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated and from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinomasbut the percentage of responders were few compared to those who did notrespond.

B. Vaccination

The invention includes the use of immunotherapy using E6 and E7 peptidesfrom HPV to induce or improve a cell-mediated immune response. This hasparticular significance for a patient who exhibits no or a low CMIresponse to E6 and/or E7 peptides from HPV. Peptides or polypeptidesthat comprises all or part of an amino acid sequence of SEQ ID NO:1 to19 may be clinically very important as an effective vaccine for both thetreatment and prevention of HPV-infection, including the prevention ofHPV-associated pre-cancerous or cancerous growths, in inducingcell-mediated immune responses in patients.

Once produced, synthesized and/or purified, the peptides andpolypeptides of the present invention may be prepared as a vaccine foradministration to a patient. It also is contemplated that the peptides,polypeptides and vaccines of the invention may be combined with othervaccines or vaccine components, such as other additional antigens, tostimulate an immune response to the antigens. In this embodiment,preferred additional antigens are those implicated as being specific orpreferentially expressed in cancers and hyperproliferative conditions.Additional antigens and vaccines that are contemplated for combinationwith the peptides, polypeptides and vaccines of the present inventionincluded those described in U.S. Pat. Nos. 5,840,317 and 5,882,654,incorporated herein by reference.

One of ordinary skill in the art would be able to envision an array ofpotential therapeutic agents and delivery protocols for testing. Forexample, the potential anti-HPV and anti-tumor agents may be naturalproducts or synthetic molecules of human design. Moreover, the modelprovides a vehicle for selection of effective agents from among abattery of known and novel compounds. The dosage and delivery mode ofany particular potential therapeutic agent can be determined on thebasis of well established guidelines for preparing pharmaceuticallyactive compositions. The test compounds may be administered, forexample, intravenously, intradermally, intramuscularly, topically,orally, or by any other pharmaceutically effective route. Using theanimals produced by the method of the present invention, an investigatorcan now, for the first time, evaluate prophylactic and therapeuticagents against high risk human papillomavirus-induced disease, possiblyincluding virus replication and transmission. These may include, amongothers, chemical-type pharmaceuticals, genetic therapies, antisenseinhibitory strategies, or prophylactic or therapeutic vaccination. Manymethods of evaluating the results of laboratory tests of proposedtherapeutics are known.

Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhold limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmetter-Guerin) and Corynebacterium parvum. In addition touse in immunotherapies of the invention, adjuvants may be used toenhance detection of a cell-mediated immune response in the context ofthe present invention.

C. Targeted Delivery Systems

To test for a virus-specific T cell response, in some embodiments of theclaimed invention, HPV polypeptides or peptides can be delivered totarget cells to express fragments of the viral protein on their surfacesfor the purpose of eliciting a T-cell response. There are variousmethods of delivery including perfusion, transfection of an expressionconstruct, viral vectors, and other means disclosed below.

1. Transfer by Perfusion

An embodiment of the claimed invention transfers peptides or acombination of peptides into cells via perfusion. Continuous perfusionof an expression construct or a viral construct also is contemplated.The amount of construct or peptide delivered in continuous perfusion canbe determined by the amount of uptake that is desirable. The presentinvention discloses an example of perfusion whereby a cell culture withan initial concentration of 10⁶ cells/ml can first be labeled, washed,and then incubated with 100 μg of synthetic peptide for two hours.

2. Expression Vectors

The delivery of therapeutic peptides can be accomplished usingexpression vectors. In the present embodiment of the invention, HPVpolypeptides and peptides are delivered to target cells through the useof expression constructs. Throughout this application, the term“expression construct” is meant to include any type of genetic constructcontaining a nucleic acid coding for an HPV polypeptide. A “viralvector” refers to an expression construct that is derived primarily fromviral sequences. In order for the construct to effect expression, thepolynucleotide encoding the HPV polynucleotide will be under thetranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the host cell, or byintroduced synthetic machinery, that is required to initiate thespecific transcription of a gene. The phrase “under transcriptionalcontrol” means that the promoter is in the correct location in relationto the polynucleotide to control RNA polymerase initiation andexpression of the polynucleotide.

The term promoter will be used herein to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anHPV polynucleotide is not believed to be critical, so long as it iscapable of expressing the polynucleotide in the targeted cell. Thus,where a human cell is targeted, it is preferable to position thepolynucleotide coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell. Generallyspeaking, such a promoter might include either a human or viralpromoter. The use of other viral or mammalian cellular or bacterialphage promoters, which are well-known in the art, to achieve expressionof polynucleotides is contemplated as well, provided that the levels ofexpression are sufficient to induce a T-cell response.

By employing a promoter with well-known properties, the level andpattern of expression of a polynucleotide following transfection can beoptimized. For example, selection of a promoter that is active inspecific cells, such as tyrosinase (melanoma), alpha-fetoprotein andalbumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression of HPVpolynucleotides. Further, selection of a promoter that is regulated inresponse to specific physiologic signals can permit inducible expressionof the HPV polypeptide construct.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are frequently overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression of anHPV polynucleotide construct. Use of a T3, T7 or SP6 cytoplasmicexpression system is another possible embodiment. Eukaryotic cells cansupport cytoplasmic transcription from certain bacteriophage promotersif the appropriate bacteriophage polymerase is provided, either as partof the delivery complex or as an additional genetic expression vector.

In certain embodiments of the invention, the delivery of an expressionvector in a cell may be identified in vitro or in vivo by including amarker in the expression vector. The marker would result in anidentifiable change to the transfected cell permitting identification ofexpression. Usually, the inclusion of a drug selection marker aids incloning and in the selection of transformants. Alternatively, enzymessuch as herpes simplex virus thymidine kinase (tk) (eukaryotic) orchloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed.Immunologic markers also can be employed. The selectable marker employedis not believed to be important, so long as it is capable of beingexpressed along with the polynucleotide encoding an HPV polypeptide.Further examples of selectable markers are well known to one of skill inthe art.

One will typically include a polyadenylation signal to effect properpolyadenylation of the transcript. The nature of the polyadenylationsignal is not believed to be crucial to the successful practice of theinvention, and any such sequence may be employed. The inventor hasemployed the SV40 polyadenylation signal in that it was convenient andknown to function well in the target cells employed. Also contemplatedas an element of the expression construct is a terminator. Theseelements can serve to enhance message levels and to minimize readthrough from the construct into other sequences.

3. Viral Vectors

In some embodiments of the present invention, an expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and, in some cases, integrate into the host cellchromosomes, have made them attractive candidates for gene transfer into mammalian cells. However, because it has been demonstrated thatdirect uptake of naked DNA, as well as receptor-mediated uptake of DNAcomplexes (discussed below), expression vectors need not be viral but,instead, may be any plasmid, cosmid, or phage construct that is capableof supporting expression of encoded genes in mammalian cells, such aspUC or Bluescript™ plasmid series.

a. Retroviruses

The retrovirus class is subdivided into three major groups: oncoviruses,such as murine leukemia virus; lentiviruses, and foamy viruses(spumaviruses). Retroviruses are single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants.

The retroviral genome contains three genes—gag, pol, and env—that encodecapsid proteins, polymerase enzyme, and envelope components,respectively. A sequence found upstream from the gag gene, termed Ψ,functions as a signal for packaging of the genome into virions. Two longterminal repeat (LTR) sequences are present at the 5′ and 3′ ends of theviral genome. These contain strong promoter and enhancer sequences andare also required for integration in the host cell genome (Coffin,1990).

In order to construct a retroviral vector, a nucleic acid encoding anHPV polypeptide is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. Alternatively, a mutated HPV virus that isincapable of leading to HPV infection can be used. In order to producevirions, a packaging cell line containing the gag, pol and env genes butwithout the LTR and Ψ components is constructed (Mann, 1983). When arecombinant plasmid containing a human cDNA, together with theretroviral LTR and Ψ sequences, is introduced into this cell line (bycalcium phosphate precipitation for example), the Ψ sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Mann, 1983). The media containing the recombinantretroviruses is then collected, optionally concentrated, and used forgene transfer. Retroviral vectors are able to infect a broad variety ofcell types. However, integration and stable expression require thedivision of host cells (Paskind, 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux, 1989).

b. Adenoviruses

Human adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 kb (Tooze, 1981). As a model system foreukaryotic gene expression, adenoviruses have been widely studied andwell characterized, which makes them an attractive system fordevelopment of adenovirus as a gene transfer system. This group ofviruses is relatively simple to grow and manipulate, and exhibits abroad host range in vitro and in vivo. In lytically infected cells,adenoviruses are capable of shutting off host protein synthesis,directing cellular machineries to synthesize large quantities of viralproteins, and producing copious amounts of virus.

The E1 region of the genome includes E1A and E1B, which encode proteinsresponsible for transcription regulation of the viral genome, as well asa few cellular genes. E2 expression, including E2A and E2B, allowssynthesis of viral replicative functions, e.g. DNA-binding protein, DNApolymerase, and a terminal protein that primes replication. E3 geneproducts prevent cytolysis by CTLs and tumor necrosis factor and appearto be important for viral propagation. Functions associated with the E4proteins include DNA replication, late gene expression, and host cellshutoff. The late gene products include most of the virion capsidproteins, and these are expressed only after most of the processing of asingle primary transcript from the major late promoter has occurred. Themajor late promoter (MLP) exhibits high efficiency during the late phaseof the infection (Stratford-Perricaudet and Perricaudet, 1991).

As only a small portion of the viral genome appears to be required incis (Tooze, 1981), adenovirus-derived vectors offer excellent potentialfor the substitution of large DNA fragments when used in connection withcell lines such as 293 cells. Ad5-transformed human embryonic kidneycell lines (Graham, 1977) have been developed to provide the essentialviral proteins in trans. The characteristics of adenoviruses render themgood candidates for use in targeting cells in vivo (Grunhaus andHorwitz, 1992).

Particular advantages of an adenovirus system for delivering foreignproteins to a cell include (i) the ability to substitute relativelylarge pieces of viral DNA by foreign DNA; (ii) the structural stabilityof recombinant adenoviruses; (iii) the safety of adenoviraladministration to humans; and (iv) lack of any known association ofadenoviral infection with cancer or malignancies; (v) the ability toobtain high titers of the recombinant virus; and (vi) the highinfectivity of adenovirus.

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus that is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Sequences encoding relatively largeforeign proteins can be expressed when additional deletions are made inthe adenovirus genome. For example, adenoviruses deleted in both E1 andE3 regions are capable of carrying up to 10 kilobases of foreign DNA andcan be grown to high titers in 293 cells (Stratford-Perricaudet andPerricaudet, 1991). Surprisingly persistent expression of transgenesfollowing adenoviral infection has also been reported.

c. AAV Vectors

Adeno-associated virus (AAV) is an attractive vector system for use inthe cell transduction of the present invention as it has a highfrequency of integration and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broadhost range for infectivity (Lebkowski, 1988; McLaughlin, 1988; Laughlin,1986; Tratschin, 1984). Details concerning the generation and use ofrAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No.4,797,368, each incorporated herein by reference.

Studies demonstrating the use of AAV in gene delivery include LaFace etal. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al.(1994). Recombinant AAV vectors have been used successfully for in vitroand in vivo transduction of marker genes (Kaplitt, 1994; Shelling andSmith, 1994; Yoder, 1994; Zhou, 1994; Samulski, 1989; Lebkowski, 1988;McLaughlin, 1988; Tratschin, 1985; Hermonat and Muzyczka, 1984) andgenes involved in human diseases (Luo, 1994; Walsh, 1994; Wei, 1994;Flotte, 1992; Ohi, 1990). Recently, an AAV vector has been approved forphase I human trials for the treatment of cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (either adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells (Muzyczka, 1992). Inthe absence of coinfection with helper virus, the wild type AAV genomeintegrates through its ends into human chromosome 19 where it resides ina latent state as a provirus (Samulski, 1991; Kotin, 1990). rAAV,however, is not restricted to chromosome 19 for integration unless theAAV Rep protein is also expressed (Shelling and Smith, 1994). When acell carrying an AAV provirus is superinfected with a helper virus, theAAV genome is “rescued” from the chromosome or from a recombinantplasmid, and a normal productive infection is established (Muzyczka,1992; Kotin, 1990; Samulski, 1989; McLaughlin, 1988).

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats (McLaughlin, 1988; Samulski, 1989; each incorporated herein byreference) and an expression plasmid containing the wild type AAV codingsequences without the terminal repeats, for example pIM45 (McCarty,1991; incorporated herein by reference). The cells are also infected ortransfected with adenovirus or plasmids carrying the adenovirus genesrequired for AAV helper function. rAAV virus stocks made in such fashionare contaminated with adenovirus which must be physically separated fromthe rAAV particles (for example, by cesium chloride densitycentrifugation). Alternatively, adenovirus vectors containing the AAVcoding regions or cell lines containing the AAV coding regions and someor all of the adenovirus helper genes could be used (Clark, 1995; Yang,1994). Cell lines carrying the rAAV DNA as an integrated provirus canalso be used (Flotte, 1995).

d. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Coupar, 1988; Ridgeway, 1988; Baichwal and Sugden, 1986), and herpesviruses may also be employed. These viruses offer several attractivefeatures for various mammalian cells (Horwich, 1990; Friedmann, 1989;Coupar, 1988; Ridgeway, 1988; Baichwal and Sugden, 1986).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. in vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich, 1990). This suggestedthat large portions of the genome could be replaced with foreign geneticmaterial. The hepatotropism and persistence (integration) wereparticularly attractive properties for liver-directed gene transfer.Chang (1991) recently introduced the chloramphenicol acetyltransferase(CAT) gene into duck hepatitis B virus genome in the place of thepolymerase, surface, and pre-surface coding sequences. It wascotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang, 1991).

e. Non-Viral Transfer Methods

Several non-viral methods for the transfer of expression vectors intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe, 1990) DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa, 1986; Potter, 1984), direct microinjection(Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene,1982; Fraley, 1979) and lipofectamine-DNA complexes, cell sonication(Fechheimer, 1987), gene bombardment using high velocitymicroprojectiles (Yang, 1990), polycations (Boussif, 1995), andreceptor-mediated transfection (Wu and Wu, 1988; Wu and Wu, 1987). Someof these techniques may be successfully adapted for in vivo or ex vivouse.

D. Colloidal Dispersion Systems

Colloidal dispersion systems constitute targeted delivery vehicles.These dispersion systems include macromolecule complexes, nanocapsulescomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al.). In addition to mammalian cells, liposomeshave been used for delivery of polynucleotides in plant, yeast andbacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high exigency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Manning, et al., Biotechniques, 6:682, 1988). The presentembodiment of the invention propose that the synthetic peptides can beformulated as a liposome. The composition of the liposome is usually acombination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

E. Pharmaceutical Compositions:

The present invention contemplates the use of the synthetic peptides inthe form of a pharmaceutical compositions. In general a pharmaceuticalcomposition will comprise an effective amount of one or moreproteinaceous sequence, nucleic acid or antibody or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of anpharmaceutical composition that contains at least one proteinaceoussequence, nucleic acid or antibody or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

The proteinaceous sequence, nucleic acid or antibody may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it need to besterile for such routes of administration as injection. The presentinvention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, rectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, topically,locally, using aerosol, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the forgoing as would be known to one ofordinary skill in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The proteinaceous sequence, nucleic acid or antibody may be formulatedinto a composition in a free base, neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts,e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic: agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the proteinaceous sequence, nucleic acid orantibody is prepared for administration by such routes as oralingestion. In these embodiments, the solid composition may comprise, forexample, solutions, suspensions, emulsions, tablets, pills, capsules(e.g., hard or soft shelled gelatin capsules), sustained releaseformulations, buccal compositions, troches, elixirs, suspensions,syrups, wafers, or combinations thereof. Oral compositions may beincorporated directly with the food of the diet. Preferred carriers fororal administration comprise inert diluents, assimilable edible carriersor combinations thereof. In other aspects of the invention, the oralcomposition may be prepared as a syrup or elixir. A syrup or elixir, andmay comprise, for example, at least one active agent, a sweeteningagent, a preservative, a flavoring agent, a dye, a preservative, orcombinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof, an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Because peptides can be administered to a patient as an immunotherapy,lipids-based compositions are relevant to the invention. These arediscussed in further detail below.

In certain embodiments, the present invention concerns a novelcomposition comprising one or more lipids associated with at least onepeptide. A lipid is a substance that is characteristically insoluble inwater and extractable with an organic solvent. Lipids include, forexample, the substances comprising the fatty droplets that naturallyoccur in the cytoplasm as well as the class of compounds which are wellknown to those of skill in the art which contain long-chain aliphatichydrocarbons and their derivatives, such as fatty acids, alcohols,amines, amino alcohols, and aldehydes. Of course, compounds other thanthose specifically described herein that are understood by one of skillin the art as lipids are also encompassed by the compositions andmethods of the present invention.

A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

A. Lipid Types

A neutral fat may comprise a glycerol and a fatty acid. A typicalglycerol is a three carbon alcohol. A fatty acid generally is a moleculecomprising a carbon chain with an acidic moeity (e.g., carboxylic acid)at an end of the chain. The carbon chain may of a fatty acid may be ofany length, however, it is preferred that the length of the carbon chainbe of from about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29, toabout 30 or more carbon atoms, and any range derivable therein. However,a preferred range is from about 14 to about 24 carbon atoms in the chainportion of the fatty acid, with about 16 to about 18 carbon atoms beingparticularly preferred in certain embodiments. In certain embodimentsthe fatty acid carbon chain may comprise an odd number of carbon atoms,however, an even number of carbon atoms in the chain may be preferred incertain embodiments. A fatty acid comprising only single bonds in itscarbon chain is called saturated, while a fatty acid comprising at leastone double bond in its chain is called unsaturated.

Specific fatty acids include, but are not limited to, linoleic acid,oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid,myristic acid, arachidic acid, palmitoleic acid, arachidonic acidricinoleic acid, tuberculosteric acid, lactobacillic acid. An acidicgroup of one or more fatty acids is covalently bonded to one or morehydroxyl groups of a glycerol. Thus, a monoglyceride comprises aglycerol and one fatty acid, a diglyceride comprises a glycerol and twofatty acids, and a triglyceride comprises a glycerol and three fattyacids.

A phospholipid generally comprises either glycerol or an sphingosinemoiety, an ionic phosphate group to produce an amphipathic compound, andone or more fatty acids. Types of phospholipids include, for example,phophoglycerides, wherein a phosphate group is linked to the firstcarbon of glycerol of a diglyceride, and sphingophospholipids (e.g.,sphingomyelin), wherein a phosphate group is esterified to a sphingosineamino alcohol. Another example of a sphingophospholipid is a sulfatide,which comprises an ionic sulfate group that makes the moleculeamphipathic. A phopholipid may, of course, comprise further chemicalgroups, such as for example, an alcohol attached to the phosphate group.Examples of such alcohol groups include serine, ethanolamine, choline,glycerol and inositol. Thus, specific phosphoglycerides include aphosphatidyl serine, a phosphatidyl ethanolamine, a phosphatidylcholine, a phosphatidyl glycerol or a phosphotidyl inositol. Otherphospholipids include a phosphatidic acid or a diacetyl phosphate. Inone aspect, a phosphatidylcholine comprises adioleoylphosphatidylcholine (a.k.a. cardiolipin), an eggphosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoylphosphatidylcholine, a monopalmitoyl phosphatidylcholine, a monostearoylphosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutroylphosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproylphosphatidylcholine, a diheptanoyl phosphatidylcholine, a dicapryloylphosphatidylcholine or a distearoyl phosphatidylcholine.

A glycolipid is related to a sphinogophospholipid, but comprises acarbohydrate group rather than a phosphate group attached to a primaryhydroxyl group of the sphingosine. A type of glycolipid called acerebroside comprises one sugar group (e.g., a glucose or galactose)attached to the primary hydroxyl group. Another example of a glycolipidis a ganglioside (e.g., a monosialoganglioside, a GM1), which comprisesabout 2, about 3, about 4, about 5, about 6, to about 7 or so sugargroups, that may be in a branched chain, attached to the primaryhydroxyl group. In other embodiments, the glycolipid is a ceramide(e.g., lactosylceramide).

A steroid is a four-membered ring system derivative of a phenanthrene.Steroids often possess regulatory functions in cells, tissues andorganisms, and include, for example, hormones and related compounds inthe progestagen (e.g. progesterone), glucocoricoid (e.g., cortisol),mineralocorticoid (e.g., aldosterone), androgen (e.g., testosterone) andestrogen (e.g., estrone) families. Cholesterol is another example of asteroid, and generally serves structural rather than regulatoryfunctions. Vitamin D is another example of a sterol, and is involved incalcium absorption from the intestine.

A terpene is a lipid comprising one or more five carbon isoprene groups.Terpenes have various biological functions, and include, for example,vitamin A, coenyzme Q and carotenoids (e.g., lycopene and β-carotene).

B. Charged and Neutral Lipid Compositions

In certain embodiments, a lipid component of a composition is unchargedor primarily uncharged. In one embodiment, a lipid component of acomposition comprises one or more neutral lipids. In another aspect, alipid component of a composition may be substantially free of anionicand cationic lipids, such as certain phospholipids (e.g., phosphatidylcholine) and cholesterol. In certain aspects, a lipid component of anuncharged or primarily uncharged lipid composition comprises about 95%,about 96%, about 97%, about 98%, about 99% or 100% lipids without acharge, substantially uncharged lipid(s), and/or a lipid mixture withequal numbers of positive and negative charges.

In other aspects, a lipid composition may be charged. For example,charged phospholipids may be used for preparing a lipid compositionaccording to the present invention and can carry a net positive chargeor a net negative charge. In a non-limiting example, diacetyl phosphatecan be employed to confer a negative charge on the lipid composition,and stearylamine can be used to confer a positive charge on the lipidcomposition.

C. Making Lipids

Lipids can be obtained from natural sources, commercial sources orchemically synthesized, as would be known to one of ordinary skill inthe art. For example, phospholipids can be from natural sources, such asegg or soybean phosphatidylcholine, brain phosphatidic acid, brain orplant phosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine. In another example, lipids suitable for useaccording to the present invention can be obtained from commercialsources. For example, dimyristyl phosphatidylcholine (“DMPC”) can beobtained from Sigma Chemical Co., dicetyl phosphate (“DCP”) is obtainedfrom K & K Laboratories (Plainview, N.Y.); cholesterol (“Chol”) isobtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol(“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc.(Birmingham, Ala.). In certain embodiments, stock solutions of lipids inchloroform or chloroform/methanol can be stored at about −20° C.Preferably, chloroform is used as the only solvent since it is morereadily evaporated than methanol.

D. Lipid Composition Structures

In a preferred embodiment of the invention, the peptide may beassociated with a lipid. A peptide associated with a lipid may bedispersed in a solution containing a lipid, dissolved with a lipid,emulsified with a lipid, mixed with a lipid, combined with a lipid,covalently bonded to a lipid, contained as a suspension in a lipid,contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure. A lipid or lipid/chimericpolypeptide associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine (Gibco BRL)-chimericpolypeptide or Superfect (Qiagen)-chimeric polypeptide complex is alsocontemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

1. Emulsions

A lipid may be comprised in an emulsion. A lipid emulsion is asubstantially permanent heterogenous liquid mixture of two or moreliquids that do not normally dissolve in each other, by mechanicalagitation or by small amounts of additional substances known asemulsifiers. Methods for preparing lipid emulsions and adding additionalcomponents are well known in the art (e.g., Modern Pharmaceutics, 1990,incorporated herein by reference).

For example, one or more lipids are added to ethanol or chloroform orany other suitable organic solvent and agitated by hand or mechanicaltechniques. The solvent is then evaporated from the mixture leaving adried glaze of lipid. The lipids are resuspended in aqueous media, suchas phosphate buffered saline, resulting in an emulsion. To achieve amore homogeneous size distribution of the emulsified lipids, the mixturemay be sonicated using conventional sonication techniques, furtheremulsified using microfluidization (using, for example, aMicrofluidizer, Newton, Mass.), and/or extruded under high pressure(such as, for example, 600 psi) using an Extruder Device (LipexBiomembranes, Vancouver, Canada).

2. Micelles

A lipid may be comprised in a micelle. A micelle is a cluster oraggregate of lipid compounds, generally in the form of a lipidmonolayer, and may be prepared using any micelle producing protocolknown to those of skill in the art (e.g., Canfield et al., 1990;El-Gorab et al, 1973; Shinoda et al., 1963; and Fendler et al., 1975,each incorporated herein by reference). For example, one or more lipidsare typically made into a suspension in an organic solvent, the solventis evaporated, the lipid is resuspended in an aqueous medium, sonicatedand then centrifuged.

3. Liposomes

In particular embodiments, a lipid comprises a liposome. A “liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes may be characterized as having vesicularstructures with a bilayer membrane, generally comprising a phospholipid,and an inner medium that generally comprises an aqueous composition.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

In certain less preferred embodiments, phospholipids from naturalsources, such as egg or soybean phosphatidylcholine, brain phosphatidicacid, brain or plant phosphatidylinositol, heart cardiolipin and plantor bacterial phosphatidylethanolamine are preferably not used as theprimary phosphatide, i.e., constituting 50% or more of the totalphosphatide composition or a liposome, because of the instability andleakiness of the resulting liposomes.

In particular embodiments, a lipid and/or chimeric polypeptide may be,for example, encapsulated in the aqueous interior of a liposome,interspersed within the lipid bilayer of a liposome, attached to aliposome via a linking molecule that is associated with both theliposome and the chimeric polypeptide, entrapped in a liposome,complexed with a liposome, etc.

a. Making Liposomes

A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill in theart. Phospholipids can form a variety of structures other than liposomeswhen dispersed in water, depending on the molar ratio of lipid to water.At low ratios the liposome is the preferred structure.

For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.), suchas for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed withthe chimeric polypeptide, and/or other component(s). Tween 20 is addedto the lipid mixture such that Tween 20 is about 5% of the composition'sweight. Excess tert-butanol is added to this mixture such that thevolume of tert-butanol is at least 95%. The mixture is vortexed, frozenin a dry ice/acetone bath and lyophilized overnight. The lyophilizedpreparation is stored at −20° C. and can be used up to three months.When required the lyophilized liposomes are reconstituted in 0.9%saline. The average diameter of the particles obtained using Tween 20for encapsulating the chimeric polypeptide is about 0.7 to about 1.0 μmin diameter.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Uster 1983, Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part). Thesemethods differ in their respective abilities to entrap aqueous materialand their respective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated additional materials, such as agents including but notlimited to hormones, drugs, nucleic acid constructs and the like, areremoved by centrifugation at 29,000×g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present invention can be a variety of sizes. In certainembodiments, the liposomes are small, e.g., less than about 100 nm,about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about50 nm in external diameter. In preparing such liposomes, any protocoldescribed herein, or as would be known to one of ordinary skill in theart may be used. Additional non-limiting examples of preparing liposomesare described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323,4,533,254, 4,162,282, 4,310,505, and 4,921,706; InternationalApplications PCT/US85/01161 and PCT/US89/05040; U.K. Patent ApplicationGB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987;Mayhew et al., 1984; Cheng et al., 1987; and Gregoriadis, 1984, eachincorporated herein by reference).

A liposome suspended in an aqueous solution is generally in the shape ofa spherical vesicle, having one or more concentric layers of lipidbilayer molecules. Each layer consists of a parallel array of moleculesrepresented by the formula XY, wherein X is a hydrophilic moiety and Yis a hydrophobic moiety. In aqueous suspension, the concentric layersare arranged such that the hydrophilic moieties tend to remain incontact with an aqueous phase and the hydrophobic regions tend toself-associate. For example, when aqueous phases are present both withinand without the liposome, the lipid molecules may form a bilayer, knownas a lamella, of the arrangement XY-YX. Aggregates of lipids may formwhen the hydrophilic and hydrophobic parts of more than one lipidmolecule become associated with each other. The size and shape of theseaggregates will depend upon many different variables, such as the natureof the solvent and the presence of other compounds in the solution.

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (II) detergent dialysis and(IV) thin film hydration. In one aspect, a contemplated method forpreparing liposomes in certain embodiments is heating sonicating, andsequential extrusion of the lipids through filters or membranes ofdecreasing pore size, thereby resulting in the formation of small,stable liposome structures. This preparation produces liposomal/chimericpolypeptide or liposomes only of appropriate and uniform size, which arestructurally stable and produce maximal activity. Such techniques arewell-known to those of skill in the art (see, for example Martin, 1990).

Once manufactured, lipid structures can be used to encapsulate compoundsthat are toxic (e.g., chemotherapeutics) or labile (e.g., nucleic acids)when in circulation. The physical characteristics of liposomes depend onpH, ionic strength and/or the presence of divalent cations. Liposomescan show low permeability to ionic and/or polar substances, but atelevated temperatures undergo a phase transition which markedly alterstheir permeability. The phase transition involves a change from aclosely packed, ordered structure, known as the gel state, to a looselypacked, less-ordered structure, known as the fluid state. This occurs ata characteristic phase-transition temperature and/or results in anincrease in permeability to ions, sugars and/or drugs. Liposomalencapsulation has resulted in a lower toxicity and a longer serumhalf-life for such compounds (Gabizon et al., 1990).

Liposomes interact with cells to deliver agents via four differentmechanisms: Endocytosis by phagocytic cells of the reticuloendothelialsystem such as macrophages and/or neutrophils; adsorption to the cellsurface, either by nonspecific weak hydrophobic and/or electrostaticforces, and/or by specific interactions with cell-surface components;fusion with the plasma cell membrane by insertion of the lipid bilayerof the liposome into the plasma membrane, with simultaneous release ofliposomal contents into the cytoplasm; and/or by transfer of liposomallipids to cellular and/or subcellular membranes, and/or vice versa,without any association of the liposome contents. Varying the liposomeformulation can alter which mechanism is operative, although more thanone may operate at the same time.

Numerous disease treatments are using lipid based gene transferstrategies to enhance conventional or establish novel therapies, inparticular therapies for treating hyperproliferative diseases. Advancesin liposome formulations have improved the efficiency of gene transferin vivo (Templeton et al., 1997) and it is contemplated that liposomesare prepared by these methods. Alternate methods of preparinglipid-based formulations for nucleic acid delivery are described (WO99/18933).

In another liposome formulation, an amphipathic vehicle called a solventdilution microcarrier (SDMC) enables integration of particular moleculesinto the bi-layer of the lipid vehicle (U.S. Pat. No. 5,879,703). TheSDMCs can be used to deliver lipopolysaccharides, polypeptides, nucleicacids and the like. Of course, any other methods of liposome preparationcan be used by the skilled artisan to obtain a desired liposomeformulation in the present invention.

b. Targeting Ligands

The targeting ligand can be either anchored in the hydrophobic portionof the complex or attached to reactive terminal groups of thehydrophilic portion of the complex. The targeting ligand can be attachedto the liposome via a linkage to a reactive group, e.g., on the distalend of the hydrophilic polymer. Preferred reactive groups include aminogroups, carboxylic groups, hydrazide groups, and thiol groups. Thecoupling of the targeting ligand to the hydrophilic polymer can beperformed by standard methods of organic chemistry that are known tothose skilled in the art. In certain embodiments, the totalconcentration of the targeting ligand can be from about 0.01 to about10% mol.

Targeting ligands are any ligand specific for a characteristic componentof the targeted region. Preferred targeting ligands include proteinssuch as polyclonal or monoclonal antibodies, antibody fragments, orchimeric antibodies, enzymes, or hormones, or sugars such as mono-,oligo- and poly-saccharides (see, Heath et al., 1986) For example,disialoganglioside GD2 is a tumor antigen that has been identifiedneuroectodermal origin tumors, such as neuroblastoma, melanoma,small-cell lung carcenoma, glioma and certain sarcomas (Mujoo et al.,1986, Schulz et al., 1984). Liposomes containing anti-disialogangliosideGD2 monoclonal antibodies have been used to aid the targeting of theliposomes to cells expressing the tumor antigen (Montaldo et al., 1999;Pagan et al., 1999). In another non-limiting example, breast andgynecological cancer antigen specific antibodies are described in U.S.Pat. No. 5,939,277, incorporated herein by reference. In a furthernon-limiting example, prostate cancer specific antibodies are disclosedin U.S. Pat. No. 6,107,090, incorporated herein by reference. Thus, itis contemplated that the antibodies described herein or as would beknown to one of ordinary skill in the art may be used to target specifictissues and cell types in combination with the compositions and methodsof the present invention. In certain embodiments of the invention,contemplated targeting ligands interact with integrins, proteoglycans,glycoproteins, receptors or transporters. Suitable ligands include anythat are specific for cells of the target organ, or for structures ofthe target organ exposed to the circulation as a result of localpathology, such as tumors.

In certain embodiments of the present invention, in order to enhance thetransduction of cells, to increase transduction of target cells, or tolimit transduction of undesired cells, antibody or cyclic peptidetargeting moieties (ligands) are associated with the lipid complex. Suchmethods are known in the art. For example, liposomes have been describedfurther that specifically target cells of the mammalian central nervoussystem (U.S. Pat. No. 5,786,214, incorporated herein by reference). Theliposomes are composed essentially ofN-glutarylphosphatidylethanolamine, cholesterol and oleic acid, whereina monoclonal antibody specific for neuroglia is conjugated to theliposomes. It is contemplated that a monoclonal antibody or antibodyfragment may be used to target delivery to specific cells, tissues, ororgans in the animal, such as for example, brain, heart, lung, liver,etc.

Still further, a chimeric polypeptide may be delivered to a target cellvia receptor-mediated delivery and/or targeting vehicles comprising alipid or liposome. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis that will be occurringin a target cell. In view of the cell type-specific distribution ofvarious receptors, this delivery method adds another degree ofspecificity to the present invention.

Thus, in certain aspects of the present invention, a ligand will bechosen to correspond to a receptor specifically expressed on the targetcell population. A cell-specific chimeric polypeptide delivery and/ortargeting vehicle may comprise a specific binding ligand in combinationwith a liposome. The chimeric polypeptide to be delivered are housedwithin a liposome and the specific binding ligand is functionallyincorporated into a liposome membrane. The liposome will thusspecifically bind to the receptor(s) of a target cell and deliver thecontents to a cell. Such systems have been shown to be functional usingsystems in which, for example, epidermal growth factor (EGF) is used inthe receptor-mediated delivery of a nucleic acid to cells that exhibitupregulation of the EGF receptor.

In certain embodiments, a receptor-mediated delivery and/or targetingvehicles comprise a cell receptor-specific ligand and a peptide Otherscomprise a cell receptor-specific ligand to which peptide to bedelivered has been operatively attached. For example, several ligandshave been used for receptor-mediated gene transfer (Wu and Wu, 1987;Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), whichestablishes the operability of the technique. In another example,specific delivery in the context of another mammalian cell type has beendescribed (Wu and Wu, 1993; incorporated herein by reference).

In still further embodiments, the specific binding ligand may compriseone or more lipids or glycoproteins that direct cell-specific binding.For example, lactosyl-ceramide, a galactose-terminal asialganglioside,have been incorporated into liposomes and observed an increase in theuptake of the insulin gene by hepatocytes (Nicolau et al., 1987). Theasialoglycoprotein, asialofetuin, which contains terminal galactosylresidues, also has been demonstrated to target liposomes to the liver(Spanjer and Scherphof, 1983; Hara et al., 1996). The sugars mannosyl,fucosyl or N-acetyl glucosamine, when coupled to the backbone of apolypeptide, bind the high affinity manose receptor (U.S. Pat. No.5,432,260, specifically incorporated herein by reference in itsentirety). It is contemplated that the cell or tissue-specifictransforming constructs of the present invention can be specificallydelivered into a target cell or tissue in a similar manner.

In another example, lactosyl ceramide, and peptides that target the LDLreceptor related proteins, such as apolipoprotein E3 (“Apo E”) have beenuseful in targeting liposomes to the liver (Spanjer and Scherphof, 1983;WO 98/0748).

Folate and the folate receptor have also been described as useful forcellular targeting (U.S. Pat. No. 5,871,727). In this example, thevitamin folate is coupled to the complex. The folate receptor has highaffinity for its ligand and is overexpressed on the surface of severalmalignant cell lines, including lung, breast and brain tumors.Anti-folate such as methotrexate may also be used as targeting ligands.Transferrin mediated delivery systems target a wide range of replicatingcells that express the transferrin receptor (Gilliland et al., 1980).

c. Liposome/Nucleic Acid Combinations

In certain embodiments, a liposome/chimeric polypeptide may comprise anucleic acid, such as, for example, an oligonucleotide, a polynucleotideor a nucleic acid construct (e.g., an expression vector). Where abacterial promoter is employed in the DNA construct that is to betransfected into eukaryotic cells, it also will be desirable to includewithin the liposome an appropriate bacterial polymerase.

It is contemplated that when the liposome/chimeric polypeptidecomposition comprises a cell or tissue specific nucleic acid, thistechnique may have applicability in the present invention. In certainembodiments, lipid-based non-viral formulations provide an alternativeto viral gene therapies. Although many cell culture studies havedocumented lipid-based non-viral gene transfer, systemic gene deliveryvia lipid-based formulations has been limited. A major limitation ofnon-viral lipid-based gene delivery is the toxicity of the cationiclipids that comprise the non-viral delivery vehicle. The in vivotoxicity of liposomes partially explains the discrepancy between invitro and in vivo gene transfer results. Another factor contributing tothis contradictory data is the difference in liposome stability in thepresence and absence of serum proteins. The interaction betweenliposomes and serum proteins has a dramatic impact on the stabilitycharacteristics of liposomes (Yang and Huang, 1997). Cationic liposomesattract and bind negatively charged serum proteins. Liposomes coated byserum proteins are either dissolved or taken up by macrophages leadingto their removal from circulation. Current in vivo liposomal deliverymethods use aerosolization, subcutaneous, intradermal, intratumoral, orintracranial injection to avoid the toxicity and stability problemsassociated with cationic lipids in the circulation. The interaction ofliposomes and plasma proteins is largely responsible for the disparitybetween the efficiency of in vitro (Felgner et al., 1987) and in vivogene transfer (Zhu et al., 1993; Philip et al., 1993; Solodin et al.,1995; Liu et al., 1995; Thierry et al., 1995; Aksentijevich et al.,1996).

d. Lipid Administration

The actual dosage amount of a lipid composition (e.g., aliposome-chimeric polypeptide) administered to a patient can bedetermined by physical and physiological factors such as body weight,severity of condition, idiopathy of the patient and on the route ofadministration. With these considerations in mind, the dosage of a lipidcomposition for a particular subject and/or course of treatment canreadily be determined.

The present invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, rectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, topically,locally and/or using aerosol, injection, infusion, continuous infusion,localized perfusion bathing target cells directly or via a catheterand/or lavage.

IX. Kits

Certain embodiments of the present invention concerns diagnostic ortherapeutic kits. The peptides may be used in the form of a kit fordetermining the possibility of development or recurrence of aprecancerous or cancerous growth in a patient with HPV. The kit maydetermine the stimulation of T-cell lymphocyte proliferation and/or anincrease in T-helper 1 cytokine production. The components of thevarious kits may be stored in suitable container means. The containermeans will generally include at least one vial, test tube, flask,bottle, syringe or other container means, into which the peptideformulation is placed, preferably, suitably allocated. The kits may alsocomprise a second container means for containing a sterile,pharmaceutically acceptable buffer or other diluent. The kits of thepresent invention may also typically include a means for containing thevials in close confinement for commercial sale, such as, e.g., injectionor blow-molded plastic containers into which the desired vials areretained.

The kit can contain reagents for detecting an interaction (detectionreagent) between a sample and an antibody. The provided reagent can beradio-, fluorescently- or enzymatically-labeled. The kit can contain aknown radiolabeled agent capable of binding or interacting with anantibody that allows a cell-mediated immune response to be detectedand/or measured. In general, these methods will include first obtaininga sample suspected of containing such a protein, peptide or antibody,contacting the sample with an antibody or peptide in accordance with thepresent invention, as the case may be, under conditions effective toallow the formation of an immunocomplex, and then detecting the presenceof the immunocomplex.

In some embodiments, one or more E6 and/or E7 peptides is includes in asuitable container. A sample can be contacted or incubated with thepeptide(s) and then the sample can be assayed for a cell-mediated immuneresponse against the peptide(s). Thus, in some embodiments, the kitcontains a non-reacting structure. In some embodiments, the non-reactingstructure may be plastic or some other synthetic material. Thenon-reacting structure can be a type of container to hold a sample, suchas a container with a well. A container with multiple wells is alsoincluded as part of the invention. In some cases, the structure is linedwith or has a membrane attached to it. It is contemplated that a welllined with a membrane can be incubated with an E6 or E7 peptide and thenassayed using the same container for a cell-mediated immune response.This can be done by employing an antibody that can be used to detect acell mediated immune response. Such antibodies include an antibody to aTH1 or TH2 cytokine, cytokine receptor, or any other receptor on a Tcell that is indicative of a cell-mediated immune response. Detectionreagents may also be included in the kit.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatograph media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), ELISPOT,indirect immunofluorescence techniques and the like. Generally,immunocomplex formation will be detected through the use of a label,such as a radiolabel or an enzyme tag (such as alkaline phosphatase,horseradish peroxidase, or the like). Of course, one may find additionaladvantages through the use of a secondary binding ligand such as asecond antibody or a biotiniavidin ligand binding arrangement, as isknown in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising a cell capable of a cell-mediated immuneresponse, as the case may be, may be employed. Samples may includecells, cell supernatants, cell suspensions, cell extracts, enzymefractions, protein extracts, or other cell-free compositions suspectedof containing a cell capable of a cell-mediated immune response.Generally speaking, kits in accordance with the present invention willinclude at least one E6 or E7 peptide and an antibody directed against aproteinaceous composition that is associated with a cell-mediated immuneresponse, together with an immunodetection reagent and a means forcontaining the antibody, peptide, and reagent. The immunodetectionreagent will typically comprise a label associated with the antibody orantigen, or associated with a secondary binding ligand. Exemplaryligands might include a secondary antibody directed against the firstantibody or antigen or a biotin or avidin (or streptavidin) ligandhaving an associated label. Of course, as noted above, a number ofexemplary labels are known in the art and all such labels may beemployed in connection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

In one embodiment, a diagnostic kit comprises probes or primers for usewith the nucleic acid detection methods. All the essential materials andreagents required for detecting peptide markers in a biological samplemay be assembled together in a kit. This generally will comprisepreselected primers for specific markers. Also included may be enzymessuitable for amplifying nucleic acids including various polymerases (RT,Taq, etc.), deoxynucleotides and buffers to provide the necessaryreaction mixture for amplification.

Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachmarker primer pair. Preferred pairs of primers for amplifying nucleicacids are selected to amplify the sequences specified in SEQ ID NO: 1-19or a complement thereof.

In another embodiment, such kits will comprise hybridization probesspecific for peptides corresponding to the sequences specified in SEQ IDNO: 1-19, or the complement thereof. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent andenzyme as well as for each hybridization probe.

In other embodiments, the present invention concerns immunodetectionkits for use with the immunodetection methods described above. As thepeptides are generally proteins, polypeptides or peptides, the peptideswill preferably be included in the kit. The immunodetection kits willthus comprise, in suitable container means, the peptide, and optionally,an immunodetection reagent.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with orlinked to the given antibody. Detectable labels that are associated withor attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

The kits may further comprise a suitably aliquoted composition of thewild-type or mutant protein, polypeptide or polypeptide, whether labeledor unlabeled, as may be used to prepare a standard curve for a detectionassay. The kits may contain antibody-label conjugates either in fullyconjugated form, in the form of intermediates, or as separate moietiesto be conjugated by the user of the kit. The components of the kits maybe packaged either in aqueous media or in lyophilized form.

Therapeutic kits of the present invention are kits comprising an peptideSEQ ID NO: 1-19. Such kits will generally contain, in suitable containermeans, a pharmaceutically acceptable formulation of a polypeptide,peptide, biological functional equivalent, immunological fragment,domain, inhibitor, antibody, gene, polynucleotide, nucleic acid,complement, or vector expressing any of the foregoing in apharmaceutically acceptable formulation. The kit may have a singlecontainer means, or it may have distinct container means for eachcompound.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The peptide compositionsmay also be formulated into a syringeable composition. In which case,the container means may itself be a syringe, pipette, or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, or even applied to and mixed withthe other components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody may be placed, and preferably, suitably aliquoted.Where, polypeptide or peptide, or a second or third binding ligand oradditional component is provided, the kit will also generally contain asecond, third or other additional container into which this ligand orcomponent may be placed. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, and anyother reagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

Irrespective of the number or type of containers, the kits of theinvention may also comprise, or be packaged with, an instrument forassisting with the injection/administration or placement of the ultimatepeptide within the body of an animal. Such an instrument may be asyringe, pipette, forceps, or any such medically approved deliveryvehicle.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Material and Methods Patients

The present embodiment of the invention comprises a study populationthat was selected from patients seen at the colposcopy clinic of TheUniversity of Texas M. D. Anderson Cancer Center. Informed consent wasobtained from the patients, and all procedures were performed accordingto an Institutional Review Board-approved protocol. The women were 17years of age or older and not pregnant with no medical history of immunedisorders. Four groups of women were identified for this study. Group 1consisted of six women without cytological or histological diagnosis ofCIN and with an HPV negative test (CIN⁽⁻⁾/HPV⁽⁻⁾). Group 2 included 31women with a histological diagnosis of CIN and HPV positive test(CIN⁽⁺⁾/HPV⁽⁺⁾). Groups 3 and 4 were selected from women who hadundergone ablative or excisional treatment for CIN at the colposcopyclinic at least 6 months before the study. The women in groups 3 and 4were (CIN⁽⁺⁾/HPV⁽⁺⁾) before CIN treatment. However, at the time ofenrollment, which was a minimum of 6 months after CIN treatment, thewomen were only assessed for disease status. Group 3 consisted of 22women without evidence of recurrence of CIN (Recur⁽⁻⁾), and group 4included 10 with histological diagnosis of recurrent CIN (Recur⁽⁺⁾). HPVpositivity was determined using the Virapap/Viratype assay (TechnologiesInc., Gaithersburg, Md.). In this protocol, the dot blot hybridizationfor HPV RNA is performed using exfoliated cervical epithelial cellsobtained with cervical swabs. The assay method involves using a³²P-labeled DNA probe-set, which identifies HPV by type: 6/11, 16/18,and 31/33/35. Cells were isolated and processed according to themanufacturer's instructions. At the time of the study, this test wasused as part of the standard care program at the colposcopy clinic. HPVpositivity was further confirmed by PCR using DNA extracted fromparaffin-embedded biopsy material as previously described (Ting et al.,1990; Schiffman et al., 1991). The consensus primers used for the PCRanalysis were derived from the L1 open reading frame of thepapillomaviruses (MY11, GCMCAGGGWCATAAYAATGG (SEQ ID NO: 23) and MY09,CGTCCMARRGGAWACTGATC (SEQ ID NO: 24); where M=A+C, R=A+G, W=A+T, Y=C+T).The HPV-16 positivity was confirmed using a specific oligonucleotideprobe CATACACCTCCAGCACCTAA (SEQ ID NO: 25). The clinicalcharacteristics, including HPV status, of the study subjects are listedin Table 2.

TABLE 2 Characteristics of the study subjects Characteristics TotalGroup 1 Group 2 Group 3 Group 4 No. of patients 69  6 31 22 10 Medianage 31 31 31 32 27 Age range 17-54 17-43 21-50 18-54 20-39 Race White 52(75.4%) 3 (50%) 26 (83.9%) 15 (68.2%) 8 (80%) Hispanic 8 (12.6%) 3 (50%)1 (3.2%) 4 (18.2%) 0 (0%) African- 8 (12.6%) 0 (0%) 3 (9.7%) 3 (13.6%) 0(0%) American Asian 1 (1.4%) 0 (0%) 1 (3.2%) 0 (0%) 0 (0%) HPV statusNegative 6 (8.7%) 6 (100%) 0 (0%) 0 (0%) 0 (0%) Positive 63 (91.3%) 0(0%) 31 (100%) 22 (100%) 10 (100%) HPV-16 57 (90.5%) 0 (0%) 31 (100%) 17(77.3%) 9 (90%) Other HPV 6 (9.5%) 0 (0%) 0 (0%) 5 (22.7%) 1 (10%) TypesInitial Diagnosis* Negative 6 (8.7%) 6 (100%) 0 (0%) 0 (0%) 0 (0%) CIN14 (5.8%) 0 (0%) 0 (0%) 4 (18.2%) 0 (0%) CIN 2 & 3 59 (85.5%) 0 (0%) 31(100%) 18 (81.8%) 10 (100%) *Note: For group 3 the diagnosis at the timeof recruitment into study was negative for CIN, while that for group 4was positive for CIN 2/3.

Peptides

Peptide sequences corresponding to the E6 and E7 oncoproteins of HPV-16were selected on the basis of the amphipathic structures and informationrelated to known T-cell epitopes described in the literature. Table 3lists the peptides used in the present study. All peptides were made asreported earlier (Sarkar et al., 1995) using the Merrifield solid-phasemethod (Merrifield, 1963) either on a modified Vega 250 automaticpeptide synthesizer (Vega Biochemicals, Tucson, Ariz.) or by the “bag”method as described by Houghten, 1985. In most of the experiments, thepurity of the peptides used was approximately 70-80% and in some of theexperiments, peptides exhibiting a purity >95% were used with identicalresults. In addition to the E6 and E7 peptides, we used a peptide fromthe c-mos protooncogene [aa 158-170, STRTPEDSNSLGT (SEQ ID NO 22)] as anegative control. Stock solutions of peptides were prepared in PBS (pH7.0) and filter sterilized.

T-Cell Proliferation Assay

Heparinized blood was collected from the study participants byvenipuncture. PBMCs were isolated by centrifugation on a Ficoll-Hypaquedensity gradient (Histopaque-1073; Sigma Chemical Co., St. Louis, Mo.).The proliferative responses of PBMC from different individuals afterstimulation with PHA, c-mos peptide, or individual E6 and E7 peptides(Table 4) were determined using the [³H]thymidine incorporation assay aspreviously described Nehete et al., 1996). Briefly, each sample wasseeded in triplicate in 96-well microtiter plates and incubated for 7days at 37° C. in a humidified 5% CO₂ atmosphere. During the final 16-18h, 1 μCi of ³[H]thymidine (6.7 Ci/mmol; ICN Biomedicals, Inc., CostaMesa, Calif.) was added. The cells were harvested onto filter strips toestimate ³[H]thymidine incorporation. The specific radioactivity ofcells treated with various additions was calculated in each case bysubtracting the counts per minute (cpm) values obtained with cellscultured in medium alone. Data from pilot experiments showed that at 5μg/ml, each peptide yields consistent levels of proliferation. Thesignificance of T-cell proliferative responses to the individual E6 andE7 peptides (in terms of stimulation index [SI]) was calculated as thefold increase of [³H]thymidine incorporation by cells exposed to thepeptide over that by the control to which no peptide was added. An SIvalue ≧3.0, which was considered a positive response, was used for allstatistical analyses to determine the significance of proliferativeresponses and the association with disease-free or disease-recurrencestatus. In all the experiments, data from triplicate samples werecomparable with a standard error of <10%. None of the women in the fourstudy groups tested showed proliferative responses specific to thecontrol c-mos peptide (SI<2.0).

TABLE 3 Amino acid sequences of the E6 and E7 peptides from HPV-16Peptide Residues Sequence E6 peptides K9L (aa 18-26) KLPQLCTEL (SEQ IDNO: 1) E101 (aa 25-34) ELQTTIHDII (SEQ ID NO: 2) C10R (aa 37-46)CVYCKQQLLR (SEQ ID NO: 3) Q15L (aa 43-57) QLLRREVYDFAFRDL (SEQ ID NO: 4)V10C (aa 49-58) VYDFAFRDLC (SEQ ID NO: 5) P9L (aa 66-74) PYAVCDKCL (SEQID NO: 6) P10I (aa 102-111) PLCDLLIRCI (SEQ ID NO: 7) Q20P (aa 97-116)QQYNKPLCDLLIRCINCQKP (SEQ ID NO: 8) R16R (aa 131-146) RWTGRCMSCCRSSRTR(SEQ ID NO: 9) G10S (aa 141-150) GRCMSCCRSS (SEQ ID NO: 10) E7 peptidesT10Q (aa 7-15) TLHEYMLELQ (SEQ ID NO: 11) M9T (aa 12-20) MLDLQPETT (SEQID NO: 12) D9L (aa 14-22) DLQPETTDL (SEQ ID NO: 13) Q19D (aa 44-62)QAEPDRAHYNIVTFCCKCD (SEQ ID NO: 14) R9F (aa 49-57) RAHYNIVTF (SEQ ID NO:15) R9V (aa 66-74) RLCVQSTHV (SEQ ID NO: 16) L9V (aa 82-90) LLMGTLGIV(SEQ ID NO: 17) G10C (aa 85-94) GTLGIVCPIC (SEQ ID NO: 18) D20C (aa75-94) DIRTLEDLLMGTLGIVCPIC (SEQ ID NO: 19) aa, amino acid

Cytokine Analysis

Cryopreserved PBMC were used for these assays. The PBMC (1×10⁵) wereincubated with various HPV peptides in RPMI-1640 medium (containing 10%fetal calf serum) in triplicate wells of 96-well round-bottom plates for48 h at 37° C. Supernatants (100 μl) were removed from each well aftercentrifugation and stored frozen at −70° C. in another 96-well plate.The plates were then thawed and the supernatants assayed for variouscytokines (IFN-γ, IL-2, IL-4, IL-10, and IL-12) using the Cytoscreenimmunoassay kits (Biosource International, Camarillo, Calif.) accordingto the manufacturer's instructions.

Statistical Analysis

Differences in the SI values between the patient groups were assessed byPearson X² and Fisher's exact tests. For the purpose of the statisticalanalysis, significant proliferative response was defined as SI≧3.0.Statistical significance was set at p<0.05.

Example 2

A total of 69 women ranging in age from 17 to 54 years (median 31 years)were enrolled in the study. Of these 69 women, 52 were white, 8 eachwere African American and Hispanic, and one was Asian (Table 2). PBMCfrom these women were analyzed for proliferative response to thesynthetic peptides corresponding with antigenic sequences of the E6 andE7 oncoproteins of HPV-16 (Table 3) (FIG. 1A).

Analyses of proliferative responses specific to various E6 and E7peptides in each of the four different groups of patients revealed thatthe majority of patients in group 3 (Recur⁽⁻⁾) exhibited positiveresponses (SI≧3.0) to all the seven E6 peptides and 7/8 E7 peptidestested (FIG. 1A). On the other hand, only 5/31 untreated patients in thegroup 2 (CIN⁽⁺⁾/HPV⁽⁺⁾) and none in the groups 1 (CIN⁽⁻⁾/HPV⁽⁻⁾) and 4(Recur⁽⁺⁾) showed responses to any of the peptides tested. This issummarized in FIG. 1B.

The relationship between proliferative response to E6 and/or E7 peptidesand post-treatment disease status in women in groups 3 and 4 arepresented in Table 4. Whereas none of the patients in group 4 (Recur⁽⁻⁾)showed response to any E6 or E7 peptide tested, in group 3 64% ofpatients had significant proliferative responses to the E6 peptides(p=0.001), 82% to the E7 peptides (p<0.001), and 86% to at least one ofthe E6 or E7 peptides (p<0.001). There was no difference inproliferative response to a common mitogen like PHA (p=0.912, data notshown) between groups 3 and 4, suggesting that there is no impairment inthe innate immune status of these patients. These results stronglysuggest a relationship of proliferative responses to synthetic peptidesfrom the E6 and E7 oncoproteins of HPV-16 and disease-free conditionafter CIN treatment.

The inventors identified higher levels of proliferative responses to twopeptides each from the E6 (Q15L and V10C) and E7 (Q19D and R9F).Representative proliferative responses, in terms of SI values, from 2patients in groups 3 and 4 to 7 synthetic peptides from the E6oncoprotein and 8 from E7 oncoprotein of HPV-16 are shown in FIG. 2.Comparison of proliferative responses to theses four peptides showedstatistically significant differences between women in groups 3 and 4(Table 5). Whereas no proliferative responses to these peptides wereobserved in group 4, in group 3 a total of 11 women exhibited responsesto peptide Q15L (p=0.006), 10 to peptide V10C (p=0.006), 13 to peptideQ19D (p=0.002), and 10 to peptide R9F (p=0.013). As seen in Table 3,nine of the 10 amino acids in the E6 peptide V10C overlap with those ofQ15L peptide. Similarly, the 9 amino acids of the E7 peptide R9F overlapwith amino acids of the Q19D peptide. Proliferative responses specificto these four peptides together could account for all the responses(19/22 women) observed in group 3 (Recur⁽⁻⁾). These results suggestthat, with respect to HPV-specific cellular immune responses, the aminoacid sequences for the Q15L and Q19D peptides within the HPV-16oncoproteins E6 and E7, respectively, may be immunodominant regions.

The inventors also tested, on a subsample of the study population,whether these peptides will also induce production of various TH1 andTH2 cytokines. Cryopreserved PBMC from 8 women in group 3 (Recur(−)),and 6 from group 4 (Recur(+)) were stimulated in vitro with peptidesQ15L and Q19D. The amounts of various TH1 cytokines (IL-2, IL-12, andIFN-□), and TH2 cytokines (IL-4 and IL-10) in the culture supernatants,after adjusting to unstimulated cultures were shown in FIG. 3. PBMC from7 of 8 (87.5%), and 5 of 8 (62.5%) women in group 3 (Recur(−)) showedproduction of IFN-□, and IL-2, respectively, in response to both Q15Land Q19D (Table 6). Additionally, IL-12 production was observed inresponse to Q15L in PBMC from 3 of 8 women in this group, whereasQ19D-mediated production of IL-12 was evident in 6 of 8 women. On theother hand, none of the PBMC from women in this group secreted IL-4 inresponse to stimulation with peptides Q15L or Q19D, and only 3 womenshowed IL-10 production in response to either of the peptides. Incontrast to women in group 3 (Recur⁽⁻⁾), women in group 4 (Recur⁽⁺⁾)predominantly showed IL-10 production (5 of 6 with Q15L, and 6 of 6 withthe Q19D). In 1 of the 6 women in this group, IL-4 production wasobserved when the PBMC were stimulated with either of the two peptidestested (Table 6). Overall, these results showed that patients in group 3(Recur⁽⁻⁾) predominantly exhibited TH1 cytokine production (IL-2, IFN-γ,and IL-12), whereas women in group 4 (Recur⁽⁺⁾), despite not exhibitingspecific proliferative responses directed against the HPV peptides,showed production of IL-10, a TH2 cytokine.

TABLE 4 Association between proliferative response to all syntheticpeptides of HPV-16 oncoproteins E6 and/or E7 and disease statusfollowing CIN treatment Group 3 Group 4 Proliferative Response(Disease-free) (recurrence) Significance E6 peptides Yes^(a) 14 (64%) 0No^(b)  8 (36%) 10 (100%) p = 0.001 E7 peptides Yes^(a) 18 (82%) 0No^(b)  4 (18%) 10 (100%) p < 0.001 Any E6 or E7 peptide Yes^(a) 19(86%) 0 No^(b)  3 (14%) 10 (100%) p < 0.001 ^(a)SI ≧ 3.0 ^(b)SI < 3.0

TABLE 5 Association between proliferative response to specific syntheticpeptides of HPV-16 oncoproteins E6 and/or E7 and disease statusfollowing CIN treatment Group 3 Group 4 (disease-free) (recurrence)Proliferative Response (n = 22) (n = 10) Significance E6 peptides Q15LYes^(a) 11 (50%) 0 No^(b) 11 (50%) 10 (100%) P = 0.006 V10C Yes^(a) 11(50%) 0 No^(b) 11 (50%) 10 (100%) P = 0.006 E7 peptides Q19D Yes^(a) 13(59%) 0 No^(b)  9 (41%) 10 (100%) P = 0.002 R9F Yes^(a) 10 (46%) 0No^(b) 12 (54%) 10 (100% P = 0.013 ^(a)SI ≧ 3.0 ^(b)SI < 3.0

TABLE 6 Cytokine production of PBMC from patients in groups 3 and 4 inresponse to stimulation with synthetic peptides from the E6 and E7oncoproteins of HPV-16^(a) Group 3 Group 4 (n = 8) (n = 6) Cytokine^(b)Q15L^(c) Q19D^(d) Q15L Q19D IFN-γ 7/8 7/8 0/6 0/6 EL-2 5/8 5/8 0/6 0/6EL-12 3/8 6/8 0/6 1/6 IL-4 0/8 0/8 1/6 1/6 IL-10 3/8 3/8 5/6 6/6^(a)Number of patients positive/number tested. ^(b)Positivity forcytokine production is based on values above the sensitivity of the testkit used for each cytokine in terms of pg/ml: IL-2 = 8.7, IFN-γ = 4.0,IL-12 = 1.0, IL-4 = 2.0, and IL-10 = 5.0. ^(c)Q15L Peptide from the E6oncoprotein of HPV-16. ^(d)Q19D Peptide from the E7 oncoprotein ofHPV-16.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references are specifically incorporated herein byreference.

-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,162,282-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,310,505-   U.S. Pat. No. 4,533,254-   U.S. Pat. No. 4,683,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,728,575-   U.S. Pat. No. 4,728,578-   U.S. Pat. No. 4,737,323-   U.S. Pat. No. 4,797,368-   U.S. Pat. No. 4,800,159-   U.S. Pat. No. 4,883,750-   U.S. Pat. No. 4,921,706-   U.S. Pat. No. 5,028,592-   U.S. Pat. No. 5,139,941-   U.S. Pat. No. 5,279,721-   U.S. Pat. No. 5,432,260-   U.S. Pat. No. 5,786,214-   U.S. Pat. No. 5,840,317-   U.S. Pat. No. 5,840,873-   U.S. Pat. No. 5,843,640-   U.S. Pat. No. 5,843,651-   U.S. Pat. No. 5,843,663-   U.S. Pat. No. 5,846,708-   U.S. Pat. No. 5,846,717-   U.S. Pat. No. 5,846,726-   U.S. Pat. No. 5,846,729-   U.S. Pat. No. 5,849,481-   U.S. Pat. No. 5,849,486-   U.S. Pat. No. 5,849,487-   U.S. Pat. No. 5,851,772-   U.S. Pat. No. 5,853,990-   U.S. Pat. No. 5,853,992-   U.S. Pat. No. 5,853,993-   U.S. Pat. No. 5,856,092-   U.S. Pat. No. 5,861,244-   U.S. Pat. No. 5,863,732-   U.S. Pat. No. 5,863,753-   U.S. Pat. No. 5,866,331-   U.S. Pat. No. 5,871,727-   U.S. Pat. No. 5,879,703-   U.S. Pat. No. 5,882,654-   U.S. Pat. No. 5,900,481-   U.S. Pat. No. 5,905,024-   U.S. Pat. No. 5,910,407-   U.S. Pat. No. 5,912,124-   U.S. Pat. No. 5,912,145-   U.S. Pat. No. 5,919,626-   U.S. Pat. No. 5,919,630-   U.S. Pat. No. 5,925,517-   U.S. Pat. No. 5,928,862-   U.S. Pat. No. 5,928,869-   U.S. Pat. No. 5,929,227-   U.S. Pat. No. 5,932,413-   U.S. Pat. No. 5,935,791-   U.S. Pat. No. 5,939,277-   U.S. Pat. No. 6,107,090-   U.S. Pat. No. 6,135,965-   U.S. Pat. No. 6,214,874-   U.S. Pat. No. 6,238,659-   U.S. Pat. No. 6,245,568-   U.S. Pat. No. 6,258,576-   EPA No. 320,308-   EPA No. 329,822-   EPO 0273085-   GB Application No. 2202328-   GB Application No. 2193095 A-   PCT Application No. PCT/US85/01161-   PCT Application No. PCT/US87/00880-   PCT Application No. PCT/US89/01025-   PCT Application No. PCT/US89/05040;-   PCT Application WO 88/10315-   PCT Application WO 89/06700-   WO 90/07641-   WO 98/0748-   WO 99/18933-   Abbondanzo, et al., Am J Clin Pathol., 93(5):698-702, 1990.-   Abe, et al., Neurosci Res., 38(4):325-9, 2000.-   Aichele, et al., J Exp Med, 171(5):1815-20, 1990.-   Aksentijevich et al., Hum Gene Ther., 7(9):1111-22, 1996.-   Allred, et al., Arch Surg. 1990 January; 125(1): 107-13. Review.-   Altman et al., Science, 274:94-96, 1996.-   Baichwal et al., “Vectors for gene transfer derived from animal DNA    viruses: Transient and stable expression of transferred genes,” In:    Gene Transfer, Kucherlapati, R., ed.,-   Bajorin, et al., J Clin Oncol., 6(5):786-92, 1988.-   Bangham, et al., J. Mol. Biol., 13:238-252, 1965.-   Barany, et al., “Solid-Phase Peptide Synthesis,” In: The Peptides:    Analysis, Synthesis, Biology, Gross and Meinhofer, eds., Academic    Press, New York, pp. 3-284, 1980.-   Bevan, et al., Nature, 342(6249):478-9, 1989.-   Birner, et al., Mod. Pathol., 14(7):702-9, 2001.-   Boussif et al., Proc. Nat'l Acad. Sci. USA, 92:7297-7301, 1995.-   Brinton, et al., In: N. Munoz, F. X. Bosch, K. V. Shah, and A.    Meheus (eds.), The Epidemiology of Cervical Cancer and Human    Papillomavirus (IARC Scientific Publications, 119), pp. 3-23. Lyon:    Oxford University Press, 1992.-   Brown, et al., Am J Vet Res., 51(9):1476-80, 1990.-   Canfield et al., Methods in Enzymology, 189, 418-422, 1990.-   Capaldi, et al., Biochem. Biophys. Res. Comm., 74(2):425-433, 1977.-   Casement, et al., Virology, 211(1):261-7, 1995-   Cason, et al., Int. J. Cancer, 50: 349-355, 1992.-   Chang et al., Hepatology, 14:134 A, 1991.-   Chen, et al., Mol. Cell. Biol., 7:2745-2752, 1987.-   Cheng, et al., Investigative Radiology, vol. 22, pp. 47-55 (1987).-   Clark et al., Human Gene Therapy, 6:1329-1341.1995-   Clave, et al., Diagn. Mol. Pathol., 9(3) 145-150, 2000.-   Clerici, et al., J. Natl. Cancer Inst., 89: 245-250, 1997.-   Coffin, In: Virology, Fields et al., eds., Raven Press, New York,    pp. 1437-1500, 1990.-   Coupar et al., Gene, 68:1-10, 1988.-   Crowe, et al., AIDS Res Hum Retroviruses, 3(2):135-45, 1987.-   da Costa, et al., Biocell, 23(1): 65-72, 1999.-   De Gruijil, et al., J. Gen. Virol. 77, 2183-2191, 1996.-   De Jager, et al., Semin Nucl Med. 1993 April; 23(2):165-79. Review.-   Deamer, et al., in Liposomes (M. Ostro, ed.), Marcel Dekker, Inc.,    New York (1983), pp. 27-52.-   Deres, et al., Nature. 1989 Nov. 30; 342(6249):561-4.-   Doolittle, et al., Methods Mol. Biol. 1999; 109:215-37. Review-   el Gorab, et al., Biochim Biophys Acta., 306(1):58-66, 1973.-   Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987.-   Felgner et al., Proc Natl Acad Sci USA., 84(21):7413-7, 1987.-   Feltkamp, et al., Eur. J. Immunol., 23: 2242-2249, 1993.-   Fendler et al., Catalysis in Micellar and Macromolecular Systems,    Academic Press, New York, 1975.-   Flotte et al., Gene Therapy, 2:29-37, 1995.-   Flotte, et al., Am. J. Respir. Cell Mol. Biol., 7:349-356, 1992.-   Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979.-   Fraley, et al., Trends Biochem. Sci., 6:77, 1981-   Friedmann, Science, 244:1275-1281, 1989.-   Frohman, PCR Protocols: A Guide To Methods and Applications,    Academic Press, New York, 1990.-   Fujishima, et al., Cytometry., 24(4):382-9, 1996.-   Gabizon et al., Cancer Res., 50(19):6371-8, 1990.-   Ghosh, et al., In: Wu G. Wu C ed., Liver diseases, targeted    diagnosis and therapy using specific receptors and ligands, New    York: Marel Dekker, pp. 87-104, 1991.-   Gilliland et al., Cancer Res., 40(10):3564-9, 1980.-   Gloeckner, et al., J. Immunol. Methods, 252(1-2):131-8, 2001.-   Gopal, Mol. Cell Biol., 5:1188-1190, 1985.-   Graham et al., J. Gen. Virol., 36:59-72, 1977.-   Graham, et al., Virology, 52:456-467, 1973.-   Gregoriadis, et al., Biochem Biophys Res Commun., 89(4):1287-1293,    1979.-   Gregoriadis, G., ed., Liposome Technology, vol. I, pp. 30-35, 51-65    and 79-107, CRC Press Inc., Boca Raton, Fla., 1984.-   Grunhaus, et al., Seminar in Virology, 3:237-252, 1992.-   Gulbis, et al., Hum Pathol. 1993 December; 24(12):1271-85. Review.-   Hamsikova, et al., J. Infect. Dis., 170: 1424-1431, 1994-   Hara et al., Biochim Biophys Acta., 1278(1):51-8, 1996-   Harland, et al., J. Cell Biol., 101: 1094-1099, 1985.-   Heath, et al., Chem. Phys. Lipids, 40:347, 1986.-   Hermonat, et al., Proc. Nat'l Acad. Sci. USA, 81:6466-6470, 1984.-   Hope et al., Biochimica et Biophysica Acta, 812: 55-65, 1985.-   Horwich et al J. Virol., 64:642-650, 1990.-   Irie, et al., Lancet, 1(8641):786-7, 1989.-   Irie, et al., Proc Natl Acad Sci USA, 83(22):8694-8, 1986.-   Jha, et al., Lancet, 341: 1116-1118, 1993.-   Kadish, et al., J. Natl. Cancer. Inst. 89:1285-1293, 1997.-   Kaplitt et al., Nature Genetics, 8:148-154, 1994-   Kast, et al., Immunol Lett., 30(2):229-32, 1991.-   Kast, et al., J. Immunotherapy, 14: 115-120, 1993.-   Kotin et al., Proc. Nat'l Acad. Sci. USA, 87:2211-2215, 1990.-   Koutsky, et al., N Engl J Med. 327(18):1272-8, 1992-   Kurman, et al., JAMA, 271:1866-1869, 1994.-   Kurman, et al., The Bethesda system for reporting cervical/vaginal    cytologic diagnoses. Definitions, criteria and explanatory notes for    terminology and specimen adequacy. New York: Springer-Verlag. pp,    30-43, 1994.-   Kwack, et al., Mol Cells, 10(5):575-8, 2000.-   LaFace et al, Viology, 162:483-486, 1988.-   Laughlin et al., J. Virol., 60:515-524, 1986.-   Lebkowski, et al., Mol. Cell. Biol., 8:3988-3996, 1988.-   Liu et al., Biochim Biophys Acta, 1240(2):277-84, 1995.-   Lorenzato et al., J. Pathol., 194(2):171-6, 2001.-   Lorincz, et al., Obstet. Gynecol., 79: 328-337, 1992.-   Lukacher, et al., J Exp Med., 160(3):814-26, 1984.-   Luo et al., Blood, 82:suppl. 1:303 A, 1994.-   Manning, et al., Biotechniques, 6:682, 1988.-   Martin et al., Nature, 345(6277):739-743, 1990.-   Mayer et al., Biochimica et Biophysica Acta, vol. 858, pp. 161-168,    1986.-   Mayhew et al., Biochimica et Biophysica Acta, vol. 775, pp. 169-174,    1984.-   Mayhew et al., Methods in Enzymology, vol. 149, pp. 64-77, 1987.-   McCarty et al., J. Virol., 65:2936-2945, 1991.-   McLaughlin et al., J. Virol., 62:1963-1973, 1988.-   Mitchell, et al., Ann N Y Acad Sci., 690:153-66, 1993.-   Mitchell, et al., J Clin Oncol., 8(5):856-69, 1990.-   Morrison, et al., Int J Cancer. 49(1):6-13, 1991-   Morton, et al., Ann Surg., 216(4):463-82, 1992.-   Morton, et al., CA Cancer J Clin., 46(4):225-44, 1996.-   Munger, et al., J. Virol., 63: 4417-4421, 1989.-   Munoz, et al., Int. J. Cancer, 52: 743-749, 1992.-   Muzyczka, N., Curr. Top. Microbiol. Immunol., 158:97-129, 1992.-   Nakagawa, et al., Clin. Diag. Lab. Immunol., 3: 205-210, 1996.-   Nakagawa, et al., J. Infect. Dis., 175: 927-931, 1997.-   Nehete et al., J. Clin. Immunol., 16:115-124, 1996.-   Nehete, et al., Cell Immunol., 160(2):217-23, 1995.-   Nicolas, et al., In: Vectors: A survey of molecular cloning vectors    and their uses, Rodriguez and Denhardt, eds., Butterworth, Stoneham,    England, pp. 494-513, 1988.-   Nicolau et al., Biochim. Biophys. Acta, 721:185-190, 1982.-   Ohi et al., Gene, 89:279-282, 1990.-   Park, et al., Asia-Oceania J. Obstet. Gynaecol., 18: 171-175, 1992.-   Parkin, et al., Int. J. Cancer, 84:827-841, 1999.-   Paskind et al., Virology, 67:242-248, 1975.-   Plenum Press, New York, pp. 117-148, 1986.-   Poijak, et al., J. Clin. Microbiol. 37:796-797, 1999.-   Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984.-   Ravindranath, et al., Int Rev Immunol, 7(4):303-29, 1991.-   Ridgeway, In: Vectors: A survey of molecular cloning vectors and    their uses, Rodriguez R. L., Denhardt D. T., eds., Butterworth,    Stoneham, England, pp. 467-492, 1988.-   Rippe et al., Mol. Cell Biol., 10:689-695, 1990.-   Rosenberg, et al., N Engl J Med., 319(25):1676-80, 1988.-   Rossen, et al., J Immunol., 135(5):3289-97, 1985.-   Roux et al., Proc. Nat'l Acad Sci. USA, 86:9079-9083, 1989.-   Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed.,    Cold Spring Harbor Laboratory, Cold Spring, Harbor, N.Y., 1989.-   Samulski et al., EMBO J., 10:3941-3950, 1991.-   Samulski et al., J. Virol., 63:3822-3828, 1989.-   Sarkar, et al., Viral Immunol., 8: 165-174, 1995.-   Sastry, et al., Vaccine., 12(14):1281-7, 1994.-   Sastry, et al., Virology, 188: 502-509, 1992.-   Seedorf, et al., EMBO J., 6: 139-144, 1987.-   Shelling, et al., Gene Therapy, 1:165-169, 1994.-   Shepherd, et al., J. Gen. Virol., 73: 1269-1274, 1992.-   Shinoda et al., Colloidal Surfactant, Academic Press, especially    “The Formation of Micelles”, Ch. 1, 1-96, 1963.-   Solodin et al., Biochemistry, 34(41):13537-44, 1995.-   Spanjer et al., Biochim Biophys Acta, 734(1):40-7, 1983.-   Stauss, et al., Proc. Natl. Acad. Sci. USA, 89: 7871-7875, 1992.-   Strang, et al., J. Gen. Virol., 71: 423-431, 1990.-   Stratford-Penicaudet, et al., In: Human Gene Transfer, O.    Cohen-Haguenauer and M. Boiron, eds., John Libbey Eurotext,    France, p. 51-61, 1991.-   Szoka, et al., Proc. Natl. Acad. Sci., 75:4194-4198, 1978.-   Tam, et al., J. Am. Chem. Soc., 105:6442, 1983.-   Templeton et al., Nat. Biotechnol., 15(7):647-52, 1997.-   Thierry et al., Proc Natl Acad Sci USA., 92(21):9742-6, 1995.-   Tindle, et al., Proc. Natl. Acad. Sci. USA, 88: 5887-5891, 1991.-   Tobery, et al., J. Immunol. Methods, 254(1-2)59-66, 2001.-   Tooze, J., ed., Molecular Biology of DNA Tumor Viruses, 2nd ed.,    Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1991.-   Townsend, et al., Cell, 44:949-968, 1986.-   Tratschin et al., Mol. Cell. Biol., 4:2072-2081, 1984.-   Tratschin et al., Mol. Cell. Biol., 5:32581-3260, 1985.-   Tsukui, et al., Cancer Res., 56: 3967-3974, 1996.-   Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986.-   Unanue, et al., FASEB J., 13:2496-502, 1989.-   Von Knebel Doeberitz, et al., Cancer Res., 48: 3780-3786, 1988.-   Wagner et al., Mol. Cell. 40: 281-286, 1999.-   Wallace, et al., Nucl. Acid Res., 9:879, 1981-   Walsh et al., Proc. Nat'l Acad. Sci. USA, 89:7257-7261, 1994.-   Wei et al., Gene Therapy, 1:261-268, 1994.-   Wettstein, et al., In: H. Pfister (ed.), Papillomaviruses and Human    Cancer, pp. 145. Florida: CRC Press, 1990.-   Wu, et al., Biochemistry, 27:887-892, 1988.-   Wu, et al., J. Biol. Chem., 262:4429-4432, 1987.-   Yang et al., J. Virol., 68:4847-4856, 1994.-   Yoder et al., Blood, 82:suppl. 1:347 A, 1994.-   Zhou et al., Exp. Hematol. (NY), 21:928-933, 1993.-   Zhou et al., J. Exp. Med, 179:1867-1875, 1994.-   Zhu et al., Chin J Biotechnol, 9(4):257-61, 1993.-   Zur Hausen, et al., Curr. Topics Microbiol. Immunol., 186:131-156,    1994.

1-41. (canceled)
 42. A method for preventing recurrence of apre-cancerous or cancerous growth in a patient infected with HPV who hasbeen treated for the growth comprising: a) identifying a patient at riskfor recurrence of HPV-associated pre-cancerous or cancerous growth; andb) administering to the patient an effective amount of at least one E6or E7 HPV peptide to induce a cell-mediated immune response against thepeptide. 43-49. (canceled)
 50. The method of claim 42, wherein thepatient is administered an effective amount of an E6 HPV peptide. 51.The method of claim 50, wherein the E6 peptide is K9L, E10I, C10R, Q15L,V10C, P9L, P10I, Q20P, R16R, or G10S.
 52. The method of claim 50,wherein the patient is administered a combination of E6 HPV peptidesselected from the group consisting of K9L, E10I, C10R, Q15L, and V10C.53. The method of claim 42, wherein the patient is administered aneffective amount of an E7 HPV peptide.
 54. The method of claim 53,wherein the E7 HPV peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C,or D20C.
 55. The method of claim 53, wherein the patient is administereda combination of E7 HPV peptides selected from the group consisting ofT10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, and D20C.
 56. The method ofclaim 42, wherein the patient is administered at least one E6 HPVpeptide and at least one E7 HPV peptide.
 57. The method of claim 42,wherein the patient has a history of a pre-cancerous growth.
 58. Themethod of claim 57, wherein the pre-cancerous growth is cervicalintraepithelial neoplasia (CIN)
 59. The method of claim 42, wherein thepatient has a history of a cancerous growth.
 60. The method of claim 59,wherein the patient has a history of cervical cancer.
 61. The method ofclaim 42, wherein the patient has undergone ablative treatment of apre-cancerous or cancerous growth in the genitourinary tract.
 62. Themethod of claim 61, wherein the ablative treatment is selected from thegroup consisting of chemotherapy, radiotherapy, hormonal therapy, genetherapy, surgery, gene therapy, and immunotherapy.
 63. The method ofclaim 42, wherein the at least one E6 or E7 HPV peptides are comprisedin a pharmaceutical composition.
 64. The method of claim 63, wherein thepharmaceutical composition further comprises an adjuvant.
 65. The methodof claim 63, wherein the pharmaceutical composition further comprises alipid.
 66. The method of claim 65, wherein the lipid is aphosphatidylglycerol, a phosphatidylcholine, a phosphatidylserine, aphosphatidylethanolamine, a sphingolipid, a cerebroside, or aganglioside.
 67. The method of claim 66, wherein the lipid is aphosphatidylglycerol.
 68. The method of claim 67, wherein thephosphatidylglycerol is diacylphosphatidyglycerol.
 69. The method ofclaim 42, wherein identifying a patient at risk for recurrence ofHPV-associated pre-cancerous or cancerous growth comprises performing aPap Smear test.
 70. The method of claim 42, wherein identifying apatient at risk for recurrence of HPV-associated pre-cancerous orcancerous growth comprises performing colposcopy on the subject.
 71. Themethod of claim 42, wherein administering to the patient an effectiveamount of at least one E6 or E7 HPV peptide comprises administering tothe patient an expression construct comprising a nucleic acid sequencecoding for at least one E6 or E7 HPV peptide wherein the nucleic acidsequence is under the transcriptional control of a promoter.
 72. Themethod of claim 71, wherein the expression construct is comprised in aviral vector.
 73. The method of claim 72, wherein the viral vector is aretroviral vector, an adenoviral vector, an adeno-associated viralvector, a vaccinia virus vector, or a herpes virus vector.
 74. Themethod of claim 71, wherein the expression construct is comprised in aliposome.
 75. The method of claim 42, wherein the at least one E6 or E7HPV peptide is administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intravaginally,rectally, topically, intratumorally, intramuscularly, subcutaneously,mucosally, orally, or topically.